Veterinary Microbiology, 22 (1990) 69-78 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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S e l e c t i v e E x t r a c t i o n of Outer-Membrane Proteins from M e m b r a n e C o m p l e x e s of Pseudomonas maltophila by C h l o r o f o r m - M e t h a n o l JAMES CHIN and YUNG DAI
Immunology, Central Veterinary Laboratory, New South Wales Department of Agriculture, Roy Watts Road, Glenfield, Sydney, N.S. W. 2167 (Australia) {Accepted 12 September 1989)
ABSTRACT Chin, J. and Dai, Y., 1990. Selective extraction of outer-membrane proteins from membrane complexes of Pseudomonas maltophila by chloroform-methanol. Vet. Microbiol., 22: 69-78. An organic phase partitioning method is described for the selective purification of outer-membrane proteins (OMPs) from the total membrane complex of the opportunistic human and sheep pathogen, Pseudomonas maltophila. SDS-PAGE analysis confirmed that OMPs purified by chloroform-methanol treatment of the total membrane complex were not only identical to OMPs extracted from outer-membrane vesicles separated by sucrose gradient density centrifugation, but also possessed little or non-detectable levels of inner-membrane contaminants. Further analysis by enzyme linked immunosorbent assay (ELISA) and immunoblotting established that OMPs extracted by organic phase partitioning with chloroform-methanol retained antigenicityand serological activity indistinguishable from OMPs that were present in outer-membrane vesicles resolved by isopycnic sucrose density centrifugation of sarkosyl-treated membrane complexes. INTRODUCTION
Pseudomonads are ubiquitous microbes and are generally considered to be opportunistic pathogens of human and animal species. Immunologically compromised patients and burn victims are particularly vulnerable to Pseudomonas infections. Pseudomonas aeruginosa and P. maltophila have been reported to play a major role in cystic fibrosis. An interesting disease syndrome attributable to Pseudomonas skin infection occurs in sheep exposed for prolonged periods to rain (Chin and Watts, 1988). Such animals develop a transient dermatitis associated with wetting of the skin. However, these conditions are extremely favourable for microbial proliferation and it has been suggested that the production of exocellular factors by these bacteria may exacerbate the dermatitic condition (Burell et al., 1982), and thus cause the formation of prominent fleece-rot lesions. The most prevalent bacterium isolated from fleecerot lesions has been reported to be P. aeruginosa and we have shown recently 0378-1135/90/$03.50
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that one of the exoproducts produced by this organism, phospholipase C, is the primary cause of dermatitis (Chin and Watts, 1988). Other reports (London and Griffith, 1984) have suggested the possible involvement of many other Pseudomonas species and it is conceivable that the early stages of fleece-rot formation may be caused by the synergistic interaction of a Pseudomonas complex. In view of this, it may be relevant to examine the serological response of fleece-rot affected sheep to outer-membrane antigens of Pseudomonas species isolated from fleece-rot lesions. To facilitate these investigations, we have selected P. maltophila as a source bacterium for the development of an improved procedure for outer-membrane protein extraction. The standard method for isolation of outer-membrane proteins (OMPs) from Gram-negative bacteria relies on the separation of inner membrane from outer-membrane vesicles by sucrose gradient centrifugation (Schnaitman, 1970). Component proteins are then released from the respective membrane fractions by treatment with detergents. A variation of the procedure utilizes sarkosyl (Filip et al., 1973) to extract inner-membrane proteins selectively from the total membrane complex followed by sucrose gradient centrifugation. Solubilized inner-membrane proteins remain at the top of the gradient while outer-membrane vesicles are sedimented to their respective buoyant densities. Ionic and non-ionic detergents do not as a rule, appear to have the ability to extract OMPs selectively from sarkosyl-resistant membrane vesicles (Moriyon and Berman, 1982). Recently, organic phase partitioning has been used successfully to extract integral hydrophobic membrane proteins from lipidrich neurological tissues (Brunden et al., 1987 ). This method may simplify the extraction of OMPs from Pseudomonas species without the need for density centrifugation. The suitability of organic phase partitioning for the extraction of OMPs from a fleece-rot isolate of P. maltophila (Holmes et al., 1979; MacDiarmid and Burrell, 1986 ), is described in this paper. It will also be demonstrated that OMPs purified by organic phase partitioning, retain both native and subunit serological activity. MATERIALS AND METHODS
Source and growth of P. maltophila The strain of P. maltophila used in the present investigation was originally isolated from a fleece-rot lesion of sheep (London and Griffith, 1984) and was kindly donated by Dr. C.J. London. The cells were grown in 250 ml broth (1% (w/v) tryptone (Oxoid), 0.5% (w/v) yeast extract (Oxoid) and 0.5% (w/v) NaC1) in a 2-1 flask which was shaken (200 oscillations min-1) overnight at 37 ° C. Bacteria were harvested by centrifugation at 5000 for 10 min at 4 ° C and washed twice with 50 ml of sterile saline (0.15 M). The yield of bacteria was usually about 2-3 g from each flask.
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Disruption of bacteria and recovery of total membrane complex Washed and pelleted bacteria were resuspended (1 g wet wt. ) in five volumes of extraction buffer (10 mM HEPES, pH 7.2 containing 10% (w/v) sucrose and 2 mM PMSF), then passed twice through a French press at a pressure of 845 kg cm -2 (Chin and Scully, 1986). The resultant crude homogenate was clarified by centrifugation at 15 000 g for 30 min. The supernatant was then carefully layered onto 5 ml of 20% (w/v) sucrose in 10 mM HEPES (pH 7.2) in a 60 Ti tube (Beckman). Ultracentrifugation for 90 min at 150 000 g resulted in the formation of a gelatinous membrane complex (35 kP) at the bottom of the tube. This complex is composed primarily of inner-and outer-membrane vesicles.
Sarkosyl or sodium lauryl sarcosinate extraction Inner-membrane proteins were extracted from the 35 kP pellet with 2% (w/v) sarkosyl in 10 mM HEPES (pH 7.2). The extract was then loaded onto a discontinuous sucrose gradient composed of successive 5-ml layers of 20, 40 and 60% (w/v) sucrose in 10 mM HEPES (pH 7.2). The samples were centrifuged for 90 min at 150 000 g in a 60 Ti rotor at 4 ° C. Sarkosyl-solubilized inner-membrane proteins remain at the top of the gradient while outer-membrane vesicles band at the 40-60% sucrose interphase. Both inner-membrane proteins and outer-membrane vesicles were recovered with the aid of a gradient fractionator and subsequently dialysed against 10 mM HEPES (pH 7.2).
Organic phase partitioning with chloroform-methanol The method of Brunden et al. (1987) was adopted with minor modifications. SDS 4% (w/v) in 10 mM HEPES (pH 7.2) was added to the 35 kP pellet and the mixture sonicated (4 × 15 s pulses) with a sonic probe at 4 ° C. The ensuing brownish sonicate was then mixed an equal volume of CHC13-MeOH (2:1 v/v) and vortexed vigorously for 2-4 min. Centrifugation at 15 000 g for 10 min resulted in the formation of a white milky band between the organic and aqueous interphase (CMx or interphase proteins). This was removed and solubilized in an equal volume of glacial acetic acid. On some occasions, the pH of the interphase proteins was adjusted upwards by dialysis against 10 mM HEPES (pH 7.2).
Production of anti-P, maltophila antisera Rabbits were v,accinated against P. maltophila by intramuscular injection with glutaraldehyde-treated bacteria (107 cfu m1-1) emulsified in an equal volume of complete Freunds adjuvant. On day 14, a similar dose of bacteria emulsified in an equal volume of incomplete Freunds adjuvant was administered subcutaneously at 6 different sites. After a further 14 days, a final injection containing 1 mg of CMx interphase proteins emulsified in incomplete Freunds adjuvant, was given intramuscularly. Blood was collected from the
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marginal ear vein before vaccination (Bleed 0) and i week after each booster injection (Bleeds 1 and 2 ). Serum from clotted blood was delipidated by centrifugation at 25 000 g for 20 rain. Each serum sample was diluted with an equal volume of T S G M (25 m M Tris-HC1, pH 7.4; 0.15 M NaC1; glycerol 50% (v/ v); merthiolate (0.001% w/v) and stored at - 20 ° C.
SDS-PAGE Electrophoresis of proteins was carried out in a mini Hoeffer gel system under reducing conditions as described by Laemmli (1970). All samples including whole bacterial cells were heated at 100 ° C for 5 min in the presence of mercaptoethanol before being loaded onto a 10% (w/v) acrylamide running gel with a 4% (w/v) stacking gel. Electrophoresis was carried out at constant 120 V until the bromophenol blue tracking dye had moved to the bottom of the gel. Immunoblots Proteins were electrophoretically transferred from the acrylamide gel to nitrocellulose in a BioRad Trans-Blot apparatus (Bio-Rad Laboratories, Richmond, CA) under conditions essentially similar to those described by Towbin et al. (1979) except that isopropanol was used instead of methanol. Immunoblots were blocked by incubation in high salt-Tween (25 mM Tris-HC1, pH 8.9; 0.15 M NaC1 and 0.5% (v/v) Tween 20), washed in TSTw (20 mM TrisHCI pH 7.4; 0.015 M MaC1 and 0.01% (v/v) Tween 20) and then reacted with rabbit antisera diluted in TSTw. Rabbit antibodies which bound to bacterial membrane antigens on the nitrocellulose were located by reaction with horseradish peroxidase conjugated protein A (HRPA, Biorad) and then detected by incubation in freshly prepared substrate (10 mM Tris-HC1 pH 7.4; 0.12 mM 3,3-diaminobenzidine; 0.03% (v/v) H202). ELISA Cell ELISA was carried out by adding 50-#1 aliquots of a suspension of P. maltophila in TSMeOH (20 mM Tris-HC1, pH 7.4; 0.015 M NaC1 and 10% (v/v) MeOH) to each well of a 96-well microtiter plate (Flow Laboratories). The concentration of the cell suspension was adjusted spectrophometrically to 0.04 A650 units. Cells were either pelleted by centrifugation at 5000 g or the microtiter plates were allowed to dry in a fan-forced oven .(Chin and Plant, 1989). Membrane vesicles and proteins were diluted in 50 mM bicarbonate buffer (pH 9.6) to 10 ttg m1-1 and 50-#1 aliquots were also delivered to each well. Coupling was allowed to proceed overnight at 4 °C and unbound sites in all wells were blocked with skim milk (2% (w/v) in TSTw) for 1 h at 37°C. The plates were washed in TSTw followed by the sequential addition of antisera, HRPA and substrate as described by Chin and Eamens (1986).
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Protein estimation The concentration of membrane proteins in various fractions was estimated by a modified Lowry procedure (Markwell et al., 1978).
RESULTS
S D S - P A GE analysis of P. maltophila membrane proteins The Coomassie-blue stained SDS-PAGE profiles of whole bacterial cell lysate; sarkosyl extracted inner membrane proteins; outer-membrane vesicles resolved by sucrose density centrifugation; and interphase proteins extracted by chloroform-methanol (CMx) phase partitioning, are depicted in Fig. 1. Polypeptides extracted by sarkosyl were very heterogenous in size distribution while the major OMPs could be resolved into four major bands corresponding to 87, 43, 24 and 23 kDa, respectively. The four major CMx protein bands were identical to the polypeptides present in the outer-membrane vesicle fraction resolved by sucrose density centrifugation. Under conditions of heavy loading outer-membrane vesicle polypeptides were contaminated by the presence of minor bands while the CMx fractions were comparatively clean. Apart from these qualitative differences, the recovery of OMPs in the CMx fraction was consistently better (4-4.5 mg g-1 wet wt.) than that obtained from outermembrane vesicles (2-2.5 mg g-1 wet wt. ).
Fig. 1. SDS-PAGE profile of Pseudomonas maltophila bacteria and membrane fractions in 10% acrylamide stained with Coomassie-blue. Whole cells (WC), inner-membrane proteins released by sarkosyl extraction of the total membrane fraction (IMP), outer-membrane vesicles resolved by isopycnic sucrose gradient centrifugation (OMV) and OMPs released by chloroform-methanol phase partitioning (CMx), were heated for 5 min at 100 °C in the presence of 2-mercaptoethanol before aliquots containing the equivalent of 10 ]lg protein were loaded onto each lane.
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ELISA reactivity of P. maltophila antigens The serological reactivity of inner-membrane proteins, outer-membrane vesicles and CMx polypeptides were compared in an ELISA test. Since the orientation and configuration of extracted membrane proteins might be different from their topography on the bacterial cell surface in situ, methanolfixed whole cells were also included in the assay to monitor antibody binding to the bacterial cell surface. As shown in Fig. 2, serum from pre-vaccinated rabbits failed to bind to any of the assay antigens. However, sera obtained 1 week after the subcutaneous booster injection, and 1 week after the CMx booster injection, showed increased antibody recognition of bacterial cell surface determinants as well as OMV and CMx. In contrast, inner-membrane proteins were significantly less immunoreactive.
Immunoblots of P. maltophila membrane antigens The identity of the reactive subunit polypeptides in outer-membrane vesicles and CMx after immunoblotting is depicted in Fig. 3. It is clear that rabbit anti-P, maltophila antibodies in Bleeds 1 and 2, recognised similar antigens in both outer-membrane vesicles and CMx fractions. While Coomassie blue stained four distinctive polypeptide bands, antibodies in serum obtained 1 week after the first booster injection preferentially bound only to the 43-kDa subunit. A new band corresponding to 32.5 kDa was recognised by Bleed 1 sera even though this was not stained prominently by Coomassie blue in SDS-PAGE gels. Bleed 1 serum antibodies also showed moderately intense binding to two 2.0.
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1,6
1.2
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0.8
< 0') 0.4 ,,-I W
O" WC
IMP
OMV
CMx
Fig. 2. E L I S A response of rabbits after vaccination with Pseudomonas maltophila antigens. Bleed 1 was collected from rabbits 1 week after a booster injection with bacterin. Bleed 2 was collected 1 week after a second booster injection with CMx proteins. Serum was diluted 1 in 500 with TSTw and assayed against WC, IMP, OMV and CMx antigens coupled to microtiter plates at a concentration of 10 ttg m l - 1.
EXTRACTIONOF OUTER-MEMBRANEPROTEINSFROMPSEUDOMONAS MALTOPHILA
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Fig. 3. Immunoblots depicting the target specificities of immune rabbit serum against polypeptides resolved by S D S - P A G E and transferred to nitrocellulose. Whole cells (WC), outer-membrane vesicles (OMV) and CMx antigens were treated as described in Fig. 1. Sera representing Bleeds 1 and 2 were collected under conditions identical to those described in Fig. 2.
additional bands at 40 and 43 kDa; and much weaker binding to the 23-kDa band. Serum from rabbits 1 week after the CMx booster, not only showed intensified binding to the same outer membrane target antigens but also recognised new specificities corresponding to 32, 29, 25, 19 and 15 kDa, respectively. The 87 and 24 kDa polypeptides visible in Coomassie blue stained gels were apparently not recognised in immunoblots by any of the rabbit anti-P, maltophila antisera. DISCUSSION
In pathogenic Gram-negative bacteria, front-line antigens recognised by the host immune system are usually those arrayed in the outer membrane, and commonly referred to as OMPs. OMPs would also be recognised by the host immune system following the administration of bacterins. Our interest in the antigenicity of OMPs from fleece-rot isolates of Pseudomonas species prompted us to select P. maltophila as a candidate bacterium for the development of a new procedure for OMP extraction. A P. maltophila bacterin preparation was used in this investigation to generate antisera for estimating the immunoreactivity of OMPs extracted by organic phase partitioning. The isolation of OMPs has traditionally relied on the lysis of bacteria by physical (French press, sonication: Chin and Scully, 1986), chemical (urea: Lohia et al., 1984) or enzymatic (lysozyme: Verstreate et al., 1982) protocols.
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The outer membrane is then separated from the cytoplasmic or inner membrane by isopycnic sucrose gradient centrifugation (Schnaitman, 1970). Depending on the bacteria species and growth conditions, this method always suffered from the possibility of inner-membrane vesicles contaminating the outer-membrane fraction and vice versa. In some instances, sucrose gradients cannot effectively resolve outer from inner membranes (Lohia et al., 1984 ). In 1973, Filip et al. reported that sodium lauryl sarcosinate was effective in selectively solubilizing inner-membrane proteins while leaving outer-membrane vesicles intact. While this proved to be an excellent procedure for extracting cytoplasmic proteins, it did not permit rapid isolation of OMPs. Although the combined Schnaitman and Filip procedures have been used widely for many bacteria species, the resulting outer-and inner-membrane proteins have been invariably contaminated with lipopolysaccharides. The intimate association between OMPs and the lipid bilayer suggested the presence of hydrophobic domains in these proteins. The use of chloroformmethanol as a solvent for solubilizing lipids should facilitate the release of hydrophobic proteins and the results presented in Fig. 1 clearly demonstrate that organic phase partitioning selectively extracted OMPs identical to those obtained by the Schnaitman-Filip procedure. Other advantages associated with the organic phase partitioning procedure include an increased yield of qualitatively cleaner OMPs and obviates the need for further isopycnic sucrose density gradient centrifugation steps. Furthermore, chloroform-methanol treatment of total membranes would remove SDS, the detergent used to solubilize the membrane complex. Subsequent dissolution of the interphase OMPs by boiling in acetic acid would result in the hydrolysis of co-purified or contaminating lipopolysaccharides. Dialysis of the mixture would eliminate lipid A contaminants resulting in a preparation with significantly reduced endotoxin content. It must be emphasised that boiling acetic acid treatment could lead to protein denaturation and hence, a degree of loss in antigenic reactivity. The concept of phase partitioning for the isolation of hydrophobic intrinsic membrane was first attempted with the nonionic detergent Triton X- 114 ( Clemetson et al., 1984) which has a low cloud point. It has since been used successfully to extract membrane proteins from the outer membranes of Mycoplasma hyorhinis (Bricker et al., 1988) and Treponema pallidum (Radolf et al., 1988). However, the cell-wall structure of these pathogens differs significantly from Gram-negative bacteria. Consequently, detergents have never been used effectively for the selective extraction of either inner and outer proteins from total membrane preparations. Experimental evidence that the CMx proteins of P. maltophila retained their immunoreactive epitopes was confirmed by ELISA and immunoblot analyses. The ELISA data in Fig. 2 demonstrate that rabbits vaccinated with P. maltophila bacterin possessed antibodies that preferentially recognised the bacterial cell surface (whole-cell assay antigen), outer-membrane vesicles and CMx
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p r o t e i n s t h a t were u n d e n a t u r e d b y h e a t or m e r c a p t o e t h a n o l t r e a t m e n t . T h e i m m u n o b l o t s p r e s e n t e d in Fig. 3 also s h o w e d t h a t s u b u n i t p o l y p e p t i d e s a f t e r h e a t a n d m e r c a p t o e t h a n o l t r e a t m e n t still r e t a i n e d t h e i r i m m u n o r e a c t i v e epitopes. T h e finding t h a t O M P s can be easily a n d rapidly p u r i f i e d b y organic p h a s e p a r t i t i o n i n g w i t h o u t loss o f biological activity clearly offers a c o n v e n i e n t a l t e r n a t i v e to existing p r o c e d u r e s for t h e i r isolation a n d use as diagnostic rea g e n t s or vaccines. T h e p r o c e d u r e has since b e e n u s e d successfully to e x t r a c t O M P s f r o m o t h e r P s e u d o m o n a s species isolated f r o m fleece-rot lesions a n d t h e i m m u n e r e s p o n s e o f t h e sheep skin to t h e s e a n t i g e n s d u r i n g fleece-rot form a t i o n is u n d e r investigation. ACKNOWLEDGEMENTS T h i s work was s u p p o r t e d b y a g r a n t ( P r o j e c t D A N 19P ) f u n d e d b y t h e Aust r a l i a n Wool C o r p o r a t i o n o n t h e r e c o m m e n d a t i o n of t h e Wool R e s e a r c h T r u s t Fund. Yung Dai is t h e h o l d e r o f a Wool R e s e a r c h T r u s t F u n d p o s t g r a d u a t e scholarship ( D A N 1 5 P ) .
REFERENCES Bricker, T.M., Boyer, M.J., Keith, J., Watson-McKown, R. and Wise, K.S., 1988. Association of lipids with integral membrane surface proteins of mycoplasma hyorhinis. Infect. Immun., 56: 295-310. Brunden, K.R., Berg, C.T. and Poduslo, J.F., 1987. Isolation of an intergral membrane glycoprotein by choroform-methanol extraction and C3-reversed phase high performance liquid chromatography. Anal. Biochem., 164: 478-481. Burrell, D.H., Merritt, J.C., Watts, J.E. and Walker, K.H., 1982. Experimental production of dermatitis in sheep with Pseudomonas aeruginosa. Aust. Vet. J., 59: 140-144. Chin, J.C. and Eamens, G.J., 1986. Immunoreactivity of fractionated antigens obtained from an arthritogenic isolate of Erysipelothrix rhusiopathiae. Aust. Vet. J., 63: 355-358. Chin, J.C. and Plant, J.W., 1989. The temporal ELISA response pf rams to Brucella ovis following experimental infection or vaccination. Res. Vet. Sci., 46: 73-78. Chin, J.C. and Scully, C., 1986. Identification of immunoreactive antigens of Brucella ovis by ELISA profiling. Res. Vet. Sci., 41: 1-6. Chin, J.C. and Watts, J.E., 1988. Biological properties of Phospholipase c purified from a fleecerot isolate of Pseudomonas aeruginosa. J. Gen. Microbiol., 134: 2567-2575. Clemetson, K.J., Bienz, B., Zahno, M.L. and Luscher, E.F., 1984. Distribution of glycoproteins and phosphoproteins in hydrophobic and hydrophilic phases in Triton X-114 phase partition. Biochim. Biophys. Acta, 778: 465-469. Filip, C., Fletcher, G., Wulf, J.L. and Earhart, C.F., 1973. Solubilization of the cytoplasmic membrane of E. coli by the ionic detergent sodium lauryl sarcosinate. J. Bacteriol., 115: 717-722. Holmes, B., Lapage, S.P. and Easterling, B.G., 1979. Distribution in clinical material and identification of Pseudomonas maltophila. J. Clin. Pathol., 32: 66-72. Laemmli, U.D., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London), 227: 680-685.
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Lohia, A., Chattergee, A.N. and Das, J., 1984. Lysis of Vibrio cholerae cell: Direct isolation of the outer membrane from whole cells by treatment with urea. J. Gen. Microbiol., 130: 2027-2033. London, C.V. and Griffith, I.P., 1984. Characterization of Pseudomonas isolated from diseased fleece. Appl. Environ. Microbiol., 47: 993-997. MacDiarmid, J.A. and Burrell, D.H., 1986. Characterization of Pseudomonas maltophiIa isolates from fleecerot. Appl. Environ. Microbiol., 51: 346-348. Markwell, M.A.K., Haas, S.M., Bieber, L.L and Tolbert, N.E., 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87: 206-210. Moriyon, I. and Berman, D.T., 1982. Effects of nonionic, ionic and dipolar ionic detergents and EDTA on the BruceUa cell envelope. J. Bacteriol., 1'52: 822-828. Radolf, J.D., Chamberlain, N.R., Clausell, A. and Norgard, M.V., 1988. Identification and localization of intergral membrane proteins of virulent Treponema paUidum by phase partitioning with the nonionic detergent Triton X-114. Infect. Immun., 56: 490-498. Schnaitman, C.A., 1970. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coll. J. Bacteriol., 104: 890-910. Towbin, H., Staehlin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76: 4350-4354. Verstreate, D.R., Creasy, M.T., Caveney, N.T., Baldwin, C.L., Blab, M.W. and Winter, A.J., 1982. Outer membrane proteins of BruceUa abortus: Isolation and characterization. Infect. Immun., 35: 979-989.
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S e l e c t i v e E x t r a c t i o n of Outer-Membrane Proteins from M e m b r a n e C o m p l e x e s of Pseudomonas maltophila by C h l o r o f o r m - M e t h a n o l JAMES CHIN and YUNG DAI
Immunology, Central Veterinary Laboratory, New South Wales Department of Agriculture, Roy Watts Road, Glenfield, Sydney, N.S. W. 2167 (Australia) {Accepted 12 September 1989)
ABSTRACT Chin, J. and Dai, Y., 1990. Selective extraction of outer-membrane proteins from membrane complexes of Pseudomonas maltophila by chloroform-methanol. Vet. Microbiol., 22: 69-78. An organic phase partitioning method is described for the selective purification of outer-membrane proteins (OMPs) from the total membrane complex of the opportunistic human and sheep pathogen, Pseudomonas maltophila. SDS-PAGE analysis confirmed that OMPs purified by chloroform-methanol treatment of the total membrane complex were not only identical to OMPs extracted from outer-membrane vesicles separated by sucrose gradient density centrifugation, but also possessed little or non-detectable levels of inner-membrane contaminants. Further analysis by enzyme linked immunosorbent assay (ELISA) and immunoblotting established that OMPs extracted by organic phase partitioning with chloroform-methanol retained antigenicityand serological activity indistinguishable from OMPs that were present in outer-membrane vesicles resolved by isopycnic sucrose density centrifugation of sarkosyl-treated membrane complexes. INTRODUCTION
Pseudomonads are ubiquitous microbes and are generally considered to be opportunistic pathogens of human and animal species. Immunologically compromised patients and burn victims are particularly vulnerable to Pseudomonas infections. Pseudomonas aeruginosa and P. maltophila have been reported to play a major role in cystic fibrosis. An interesting disease syndrome attributable to Pseudomonas skin infection occurs in sheep exposed for prolonged periods to rain (Chin and Watts, 1988). Such animals develop a transient dermatitis associated with wetting of the skin. However, these conditions are extremely favourable for microbial proliferation and it has been suggested that the production of exocellular factors by these bacteria may exacerbate the dermatitic condition (Burell et al., 1982), and thus cause the formation of prominent fleece-rot lesions. The most prevalent bacterium isolated from fleecerot lesions has been reported to be P. aeruginosa and we have shown recently 0378-1135/90/$03.50
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that one of the exoproducts produced by this organism, phospholipase C, is the primary cause of dermatitis (Chin and Watts, 1988). Other reports (London and Griffith, 1984) have suggested the possible involvement of many other Pseudomonas species and it is conceivable that the early stages of fleece-rot formation may be caused by the synergistic interaction of a Pseudomonas complex. In view of this, it may be relevant to examine the serological response of fleece-rot affected sheep to outer-membrane antigens of Pseudomonas species isolated from fleece-rot lesions. To facilitate these investigations, we have selected P. maltophila as a source bacterium for the development of an improved procedure for outer-membrane protein extraction. The standard method for isolation of outer-membrane proteins (OMPs) from Gram-negative bacteria relies on the separation of inner membrane from outer-membrane vesicles by sucrose gradient centrifugation (Schnaitman, 1970). Component proteins are then released from the respective membrane fractions by treatment with detergents. A variation of the procedure utilizes sarkosyl (Filip et al., 1973) to extract inner-membrane proteins selectively from the total membrane complex followed by sucrose gradient centrifugation. Solubilized inner-membrane proteins remain at the top of the gradient while outer-membrane vesicles are sedimented to their respective buoyant densities. Ionic and non-ionic detergents do not as a rule, appear to have the ability to extract OMPs selectively from sarkosyl-resistant membrane vesicles (Moriyon and Berman, 1982). Recently, organic phase partitioning has been used successfully to extract integral hydrophobic membrane proteins from lipidrich neurological tissues (Brunden et al., 1987 ). This method may simplify the extraction of OMPs from Pseudomonas species without the need for density centrifugation. The suitability of organic phase partitioning for the extraction of OMPs from a fleece-rot isolate of P. maltophila (Holmes et al., 1979; MacDiarmid and Burrell, 1986 ), is described in this paper. It will also be demonstrated that OMPs purified by organic phase partitioning, retain both native and subunit serological activity. MATERIALS AND METHODS
Source and growth of P. maltophila The strain of P. maltophila used in the present investigation was originally isolated from a fleece-rot lesion of sheep (London and Griffith, 1984) and was kindly donated by Dr. C.J. London. The cells were grown in 250 ml broth (1% (w/v) tryptone (Oxoid), 0.5% (w/v) yeast extract (Oxoid) and 0.5% (w/v) NaC1) in a 2-1 flask which was shaken (200 oscillations min-1) overnight at 37 ° C. Bacteria were harvested by centrifugation at 5000 for 10 min at 4 ° C and washed twice with 50 ml of sterile saline (0.15 M). The yield of bacteria was usually about 2-3 g from each flask.
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Disruption of bacteria and recovery of total membrane complex Washed and pelleted bacteria were resuspended (1 g wet wt. ) in five volumes of extraction buffer (10 mM HEPES, pH 7.2 containing 10% (w/v) sucrose and 2 mM PMSF), then passed twice through a French press at a pressure of 845 kg cm -2 (Chin and Scully, 1986). The resultant crude homogenate was clarified by centrifugation at 15 000 g for 30 min. The supernatant was then carefully layered onto 5 ml of 20% (w/v) sucrose in 10 mM HEPES (pH 7.2) in a 60 Ti tube (Beckman). Ultracentrifugation for 90 min at 150 000 g resulted in the formation of a gelatinous membrane complex (35 kP) at the bottom of the tube. This complex is composed primarily of inner-and outer-membrane vesicles.
Sarkosyl or sodium lauryl sarcosinate extraction Inner-membrane proteins were extracted from the 35 kP pellet with 2% (w/v) sarkosyl in 10 mM HEPES (pH 7.2). The extract was then loaded onto a discontinuous sucrose gradient composed of successive 5-ml layers of 20, 40 and 60% (w/v) sucrose in 10 mM HEPES (pH 7.2). The samples were centrifuged for 90 min at 150 000 g in a 60 Ti rotor at 4 ° C. Sarkosyl-solubilized inner-membrane proteins remain at the top of the gradient while outer-membrane vesicles band at the 40-60% sucrose interphase. Both inner-membrane proteins and outer-membrane vesicles were recovered with the aid of a gradient fractionator and subsequently dialysed against 10 mM HEPES (pH 7.2).
Organic phase partitioning with chloroform-methanol The method of Brunden et al. (1987) was adopted with minor modifications. SDS 4% (w/v) in 10 mM HEPES (pH 7.2) was added to the 35 kP pellet and the mixture sonicated (4 × 15 s pulses) with a sonic probe at 4 ° C. The ensuing brownish sonicate was then mixed an equal volume of CHC13-MeOH (2:1 v/v) and vortexed vigorously for 2-4 min. Centrifugation at 15 000 g for 10 min resulted in the formation of a white milky band between the organic and aqueous interphase (CMx or interphase proteins). This was removed and solubilized in an equal volume of glacial acetic acid. On some occasions, the pH of the interphase proteins was adjusted upwards by dialysis against 10 mM HEPES (pH 7.2).
Production of anti-P, maltophila antisera Rabbits were v,accinated against P. maltophila by intramuscular injection with glutaraldehyde-treated bacteria (107 cfu m1-1) emulsified in an equal volume of complete Freunds adjuvant. On day 14, a similar dose of bacteria emulsified in an equal volume of incomplete Freunds adjuvant was administered subcutaneously at 6 different sites. After a further 14 days, a final injection containing 1 mg of CMx interphase proteins emulsified in incomplete Freunds adjuvant, was given intramuscularly. Blood was collected from the
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marginal ear vein before vaccination (Bleed 0) and i week after each booster injection (Bleeds 1 and 2 ). Serum from clotted blood was delipidated by centrifugation at 25 000 g for 20 rain. Each serum sample was diluted with an equal volume of T S G M (25 m M Tris-HC1, pH 7.4; 0.15 M NaC1; glycerol 50% (v/ v); merthiolate (0.001% w/v) and stored at - 20 ° C.
SDS-PAGE Electrophoresis of proteins was carried out in a mini Hoeffer gel system under reducing conditions as described by Laemmli (1970). All samples including whole bacterial cells were heated at 100 ° C for 5 min in the presence of mercaptoethanol before being loaded onto a 10% (w/v) acrylamide running gel with a 4% (w/v) stacking gel. Electrophoresis was carried out at constant 120 V until the bromophenol blue tracking dye had moved to the bottom of the gel. Immunoblots Proteins were electrophoretically transferred from the acrylamide gel to nitrocellulose in a BioRad Trans-Blot apparatus (Bio-Rad Laboratories, Richmond, CA) under conditions essentially similar to those described by Towbin et al. (1979) except that isopropanol was used instead of methanol. Immunoblots were blocked by incubation in high salt-Tween (25 mM Tris-HC1, pH 8.9; 0.15 M NaC1 and 0.5% (v/v) Tween 20), washed in TSTw (20 mM TrisHCI pH 7.4; 0.015 M MaC1 and 0.01% (v/v) Tween 20) and then reacted with rabbit antisera diluted in TSTw. Rabbit antibodies which bound to bacterial membrane antigens on the nitrocellulose were located by reaction with horseradish peroxidase conjugated protein A (HRPA, Biorad) and then detected by incubation in freshly prepared substrate (10 mM Tris-HC1 pH 7.4; 0.12 mM 3,3-diaminobenzidine; 0.03% (v/v) H202). ELISA Cell ELISA was carried out by adding 50-#1 aliquots of a suspension of P. maltophila in TSMeOH (20 mM Tris-HC1, pH 7.4; 0.015 M NaC1 and 10% (v/v) MeOH) to each well of a 96-well microtiter plate (Flow Laboratories). The concentration of the cell suspension was adjusted spectrophometrically to 0.04 A650 units. Cells were either pelleted by centrifugation at 5000 g or the microtiter plates were allowed to dry in a fan-forced oven .(Chin and Plant, 1989). Membrane vesicles and proteins were diluted in 50 mM bicarbonate buffer (pH 9.6) to 10 ttg m1-1 and 50-#1 aliquots were also delivered to each well. Coupling was allowed to proceed overnight at 4 °C and unbound sites in all wells were blocked with skim milk (2% (w/v) in TSTw) for 1 h at 37°C. The plates were washed in TSTw followed by the sequential addition of antisera, HRPA and substrate as described by Chin and Eamens (1986).
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Protein estimation The concentration of membrane proteins in various fractions was estimated by a modified Lowry procedure (Markwell et al., 1978).
RESULTS
S D S - P A GE analysis of P. maltophila membrane proteins The Coomassie-blue stained SDS-PAGE profiles of whole bacterial cell lysate; sarkosyl extracted inner membrane proteins; outer-membrane vesicles resolved by sucrose density centrifugation; and interphase proteins extracted by chloroform-methanol (CMx) phase partitioning, are depicted in Fig. 1. Polypeptides extracted by sarkosyl were very heterogenous in size distribution while the major OMPs could be resolved into four major bands corresponding to 87, 43, 24 and 23 kDa, respectively. The four major CMx protein bands were identical to the polypeptides present in the outer-membrane vesicle fraction resolved by sucrose density centrifugation. Under conditions of heavy loading outer-membrane vesicle polypeptides were contaminated by the presence of minor bands while the CMx fractions were comparatively clean. Apart from these qualitative differences, the recovery of OMPs in the CMx fraction was consistently better (4-4.5 mg g-1 wet wt.) than that obtained from outermembrane vesicles (2-2.5 mg g-1 wet wt. ).
Fig. 1. SDS-PAGE profile of Pseudomonas maltophila bacteria and membrane fractions in 10% acrylamide stained with Coomassie-blue. Whole cells (WC), inner-membrane proteins released by sarkosyl extraction of the total membrane fraction (IMP), outer-membrane vesicles resolved by isopycnic sucrose gradient centrifugation (OMV) and OMPs released by chloroform-methanol phase partitioning (CMx), were heated for 5 min at 100 °C in the presence of 2-mercaptoethanol before aliquots containing the equivalent of 10 ]lg protein were loaded onto each lane.
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ELISA reactivity of P. maltophila antigens The serological reactivity of inner-membrane proteins, outer-membrane vesicles and CMx polypeptides were compared in an ELISA test. Since the orientation and configuration of extracted membrane proteins might be different from their topography on the bacterial cell surface in situ, methanolfixed whole cells were also included in the assay to monitor antibody binding to the bacterial cell surface. As shown in Fig. 2, serum from pre-vaccinated rabbits failed to bind to any of the assay antigens. However, sera obtained 1 week after the subcutaneous booster injection, and 1 week after the CMx booster injection, showed increased antibody recognition of bacterial cell surface determinants as well as OMV and CMx. In contrast, inner-membrane proteins were significantly less immunoreactive.
Immunoblots of P. maltophila membrane antigens The identity of the reactive subunit polypeptides in outer-membrane vesicles and CMx after immunoblotting is depicted in Fig. 3. It is clear that rabbit anti-P, maltophila antibodies in Bleeds 1 and 2, recognised similar antigens in both outer-membrane vesicles and CMx fractions. While Coomassie blue stained four distinctive polypeptide bands, antibodies in serum obtained 1 week after the first booster injection preferentially bound only to the 43-kDa subunit. A new band corresponding to 32.5 kDa was recognised by Bleed 1 sera even though this was not stained prominently by Coomassie blue in SDS-PAGE gels. Bleed 1 serum antibodies also showed moderately intense binding to two 2.0.
E ¢. ¢q,
1,6
1.2
©
0.8
< 0') 0.4 ,,-I W
O" WC
IMP
OMV
CMx
Fig. 2. E L I S A response of rabbits after vaccination with Pseudomonas maltophila antigens. Bleed 1 was collected from rabbits 1 week after a booster injection with bacterin. Bleed 2 was collected 1 week after a second booster injection with CMx proteins. Serum was diluted 1 in 500 with TSTw and assayed against WC, IMP, OMV and CMx antigens coupled to microtiter plates at a concentration of 10 ttg m l - 1.
EXTRACTIONOF OUTER-MEMBRANEPROTEINSFROMPSEUDOMONAS MALTOPHILA
o
75
X~
.92 6,6 -45 • 31 • 21 -14
Fig. 3. Immunoblots depicting the target specificities of immune rabbit serum against polypeptides resolved by S D S - P A G E and transferred to nitrocellulose. Whole cells (WC), outer-membrane vesicles (OMV) and CMx antigens were treated as described in Fig. 1. Sera representing Bleeds 1 and 2 were collected under conditions identical to those described in Fig. 2.
additional bands at 40 and 43 kDa; and much weaker binding to the 23-kDa band. Serum from rabbits 1 week after the CMx booster, not only showed intensified binding to the same outer membrane target antigens but also recognised new specificities corresponding to 32, 29, 25, 19 and 15 kDa, respectively. The 87 and 24 kDa polypeptides visible in Coomassie blue stained gels were apparently not recognised in immunoblots by any of the rabbit anti-P, maltophila antisera. DISCUSSION
In pathogenic Gram-negative bacteria, front-line antigens recognised by the host immune system are usually those arrayed in the outer membrane, and commonly referred to as OMPs. OMPs would also be recognised by the host immune system following the administration of bacterins. Our interest in the antigenicity of OMPs from fleece-rot isolates of Pseudomonas species prompted us to select P. maltophila as a candidate bacterium for the development of a new procedure for OMP extraction. A P. maltophila bacterin preparation was used in this investigation to generate antisera for estimating the immunoreactivity of OMPs extracted by organic phase partitioning. The isolation of OMPs has traditionally relied on the lysis of bacteria by physical (French press, sonication: Chin and Scully, 1986), chemical (urea: Lohia et al., 1984) or enzymatic (lysozyme: Verstreate et al., 1982) protocols.
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The outer membrane is then separated from the cytoplasmic or inner membrane by isopycnic sucrose gradient centrifugation (Schnaitman, 1970). Depending on the bacteria species and growth conditions, this method always suffered from the possibility of inner-membrane vesicles contaminating the outer-membrane fraction and vice versa. In some instances, sucrose gradients cannot effectively resolve outer from inner membranes (Lohia et al., 1984 ). In 1973, Filip et al. reported that sodium lauryl sarcosinate was effective in selectively solubilizing inner-membrane proteins while leaving outer-membrane vesicles intact. While this proved to be an excellent procedure for extracting cytoplasmic proteins, it did not permit rapid isolation of OMPs. Although the combined Schnaitman and Filip procedures have been used widely for many bacteria species, the resulting outer-and inner-membrane proteins have been invariably contaminated with lipopolysaccharides. The intimate association between OMPs and the lipid bilayer suggested the presence of hydrophobic domains in these proteins. The use of chloroformmethanol as a solvent for solubilizing lipids should facilitate the release of hydrophobic proteins and the results presented in Fig. 1 clearly demonstrate that organic phase partitioning selectively extracted OMPs identical to those obtained by the Schnaitman-Filip procedure. Other advantages associated with the organic phase partitioning procedure include an increased yield of qualitatively cleaner OMPs and obviates the need for further isopycnic sucrose density gradient centrifugation steps. Furthermore, chloroform-methanol treatment of total membranes would remove SDS, the detergent used to solubilize the membrane complex. Subsequent dissolution of the interphase OMPs by boiling in acetic acid would result in the hydrolysis of co-purified or contaminating lipopolysaccharides. Dialysis of the mixture would eliminate lipid A contaminants resulting in a preparation with significantly reduced endotoxin content. It must be emphasised that boiling acetic acid treatment could lead to protein denaturation and hence, a degree of loss in antigenic reactivity. The concept of phase partitioning for the isolation of hydrophobic intrinsic membrane was first attempted with the nonionic detergent Triton X- 114 ( Clemetson et al., 1984) which has a low cloud point. It has since been used successfully to extract membrane proteins from the outer membranes of Mycoplasma hyorhinis (Bricker et al., 1988) and Treponema pallidum (Radolf et al., 1988). However, the cell-wall structure of these pathogens differs significantly from Gram-negative bacteria. Consequently, detergents have never been used effectively for the selective extraction of either inner and outer proteins from total membrane preparations. Experimental evidence that the CMx proteins of P. maltophila retained their immunoreactive epitopes was confirmed by ELISA and immunoblot analyses. The ELISA data in Fig. 2 demonstrate that rabbits vaccinated with P. maltophila bacterin possessed antibodies that preferentially recognised the bacterial cell surface (whole-cell assay antigen), outer-membrane vesicles and CMx
EXTRACTION OF OUTER-MEMBRANE PROTEINS FROM PSEUDOMONAS MALTOPHILA
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p r o t e i n s t h a t were u n d e n a t u r e d b y h e a t or m e r c a p t o e t h a n o l t r e a t m e n t . T h e i m m u n o b l o t s p r e s e n t e d in Fig. 3 also s h o w e d t h a t s u b u n i t p o l y p e p t i d e s a f t e r h e a t a n d m e r c a p t o e t h a n o l t r e a t m e n t still r e t a i n e d t h e i r i m m u n o r e a c t i v e epitopes. T h e finding t h a t O M P s can be easily a n d rapidly p u r i f i e d b y organic p h a s e p a r t i t i o n i n g w i t h o u t loss o f biological activity clearly offers a c o n v e n i e n t a l t e r n a t i v e to existing p r o c e d u r e s for t h e i r isolation a n d use as diagnostic rea g e n t s or vaccines. T h e p r o c e d u r e has since b e e n u s e d successfully to e x t r a c t O M P s f r o m o t h e r P s e u d o m o n a s species isolated f r o m fleece-rot lesions a n d t h e i m m u n e r e s p o n s e o f t h e sheep skin to t h e s e a n t i g e n s d u r i n g fleece-rot form a t i o n is u n d e r investigation. ACKNOWLEDGEMENTS T h i s work was s u p p o r t e d b y a g r a n t ( P r o j e c t D A N 19P ) f u n d e d b y t h e Aust r a l i a n Wool C o r p o r a t i o n o n t h e r e c o m m e n d a t i o n of t h e Wool R e s e a r c h T r u s t Fund. Yung Dai is t h e h o l d e r o f a Wool R e s e a r c h T r u s t F u n d p o s t g r a d u a t e scholarship ( D A N 1 5 P ) .
REFERENCES Bricker, T.M., Boyer, M.J., Keith, J., Watson-McKown, R. and Wise, K.S., 1988. Association of lipids with integral membrane surface proteins of mycoplasma hyorhinis. Infect. Immun., 56: 295-310. Brunden, K.R., Berg, C.T. and Poduslo, J.F., 1987. Isolation of an intergral membrane glycoprotein by choroform-methanol extraction and C3-reversed phase high performance liquid chromatography. Anal. Biochem., 164: 478-481. Burrell, D.H., Merritt, J.C., Watts, J.E. and Walker, K.H., 1982. Experimental production of dermatitis in sheep with Pseudomonas aeruginosa. Aust. Vet. J., 59: 140-144. Chin, J.C. and Eamens, G.J., 1986. Immunoreactivity of fractionated antigens obtained from an arthritogenic isolate of Erysipelothrix rhusiopathiae. Aust. Vet. J., 63: 355-358. Chin, J.C. and Plant, J.W., 1989. The temporal ELISA response pf rams to Brucella ovis following experimental infection or vaccination. Res. Vet. Sci., 46: 73-78. Chin, J.C. and Scully, C., 1986. Identification of immunoreactive antigens of Brucella ovis by ELISA profiling. Res. Vet. Sci., 41: 1-6. Chin, J.C. and Watts, J.E., 1988. Biological properties of Phospholipase c purified from a fleecerot isolate of Pseudomonas aeruginosa. J. Gen. Microbiol., 134: 2567-2575. Clemetson, K.J., Bienz, B., Zahno, M.L. and Luscher, E.F., 1984. Distribution of glycoproteins and phosphoproteins in hydrophobic and hydrophilic phases in Triton X-114 phase partition. Biochim. Biophys. Acta, 778: 465-469. Filip, C., Fletcher, G., Wulf, J.L. and Earhart, C.F., 1973. Solubilization of the cytoplasmic membrane of E. coli by the ionic detergent sodium lauryl sarcosinate. J. Bacteriol., 115: 717-722. Holmes, B., Lapage, S.P. and Easterling, B.G., 1979. Distribution in clinical material and identification of Pseudomonas maltophila. J. Clin. Pathol., 32: 66-72. Laemmli, U.D., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London), 227: 680-685.
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Lohia, A., Chattergee, A.N. and Das, J., 1984. Lysis of Vibrio cholerae cell: Direct isolation of the outer membrane from whole cells by treatment with urea. J. Gen. Microbiol., 130: 2027-2033. London, C.V. and Griffith, I.P., 1984. Characterization of Pseudomonas isolated from diseased fleece. Appl. Environ. Microbiol., 47: 993-997. MacDiarmid, J.A. and Burrell, D.H., 1986. Characterization of Pseudomonas maltophiIa isolates from fleecerot. Appl. Environ. Microbiol., 51: 346-348. Markwell, M.A.K., Haas, S.M., Bieber, L.L and Tolbert, N.E., 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87: 206-210. Moriyon, I. and Berman, D.T., 1982. Effects of nonionic, ionic and dipolar ionic detergents and EDTA on the BruceUa cell envelope. J. Bacteriol., 1'52: 822-828. Radolf, J.D., Chamberlain, N.R., Clausell, A. and Norgard, M.V., 1988. Identification and localization of intergral membrane proteins of virulent Treponema paUidum by phase partitioning with the nonionic detergent Triton X-114. Infect. Immun., 56: 490-498. Schnaitman, C.A., 1970. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coll. J. Bacteriol., 104: 890-910. Towbin, H., Staehlin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76: 4350-4354. Verstreate, D.R., Creasy, M.T., Caveney, N.T., Baldwin, C.L., Blab, M.W. and Winter, A.J., 1982. Outer membrane proteins of BruceUa abortus: Isolation and characterization. Infect. Immun., 35: 979-989.
