Vol. 39, No. 1INFECTION AND IMMUNITY, Jan. 1983, p. 394-402001 9-9567/83/010394-09$02.00/0Copyright © 1983, American Society for Microbiology

Isolation, Purification, and Partial Characterization of Brucellaabortus Matrix Protein

IGNACIO MORIYON AND DAVID T. BERMAN*

Departments of Bacteriology and Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin53706

Received 7 July 1982/Accepted 14 October 1982

Peptidoglycan sacculi with peptidoglycan-associated proteins were preparedfrom cell envelopes of Brlcella abortlus by extraction with sodium dodecyl sulfate(SDS) at 50°C. On extraction of these preparations with SDS at 100°C, a proteinwas obtained whose removal from peptidoglycan was confirmed by electronmicroscopy. Incubation of the 50°C SDS-extracted cell envelopes with 50 mMMgCl2 in SDS-2-,3-mercaptoethanol at 37°C also extracted the protein, along withlipopolysaccharide. At temperatures below 60°C, the protein did not bind SDSstrongly and had an apparent molecular weight greater than 92,000 in SDS-polyacrylamide gel electrophoresis. At higher temperatures, SDS bound strongly,and the apparent molecular weight was 38,000. Urea at 5 M did not alter theelectrophoretic mobility of this 38,000-molecular-weight form. Immunoelectro-phoresis in detergents with antisera to cell envelopes, carbohydrate staining ofSDS-polyacrylamide gels, and production of anti-lipopolysaccharide antibodiesby mice immunized with the purified protein indicated that lipopolysaccharidewas present in free and protein-bound forms. Sequential gel filtration in SDS-EDTA and SDS-NaCl removed most lipopolysaccharide. After further purifica-tion by preparative SDS-polyacrylamide gel electrophoresis, a gas-liquid chro-matographic analysis showed residual lipid tightly associated with the protein.The results suggested that the interactions between matrix proteins and otherouter membrane components are stronger in B. abo1rtis than in Escherichhia coli,which was used as a control throughout.

Extensive work on the major outer membraneproteins of gram-negative bacteria, primarilyEnterobacteriaceae (6, 33), has shown them toinclude a set of proteins with molecular weights(MWg) of about 40,000 (40K) that are tightlyassociated with the peptidoglycan (36). Becauseof their regular arrangement on the peptidogly-can, these proteins are often referred to asmatrix proteins (36, 39). They constitute thediffusion pores through which hydrophilic sub-stances of low MW penetrate the outer mem-brane (26, 27) and are essential for assembly ofthe outer membrane (29, 44). Analogous pro-teins have been described for other gram-nega-tive bacteria (14, 21, 23).There has been relatively little work on the

outer membrane proteins of Briucella spp. Du-bray and Bezard (8) have isolated three proteinsof MW 37K, 25K, and 15K by preparativesodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) of lysozyme-digestedpeptidoglycan sacculi of Brlucella abo-rtuis thathad been prepared by extracting the cell enve-lopes with SDS at 100°C and found them to beprotective immunogens in a mouse model for

brucellosis. Verstreate et al. (42) have identifiedthree clusters of proteins in the outer membraneof B. abortus, two of which (MWs of43K to 41Kand 30K) could be isolated by ion-exchangechromatography after extraction of lysozyme-digested cell envelopes with the dipolar ionicdetergent Zwittergent 314 (Calbiochem, La Jol-la, Calif.). In a preliminary report (I. Moriyonand D. T. Berman, Abstr. Annu. Meet. Am.Soc. Microbiol. 1981, K171, p. 166), we indicat-ed that a 38K protein of cell envelopes of B.abortlus behaved as a peptidoglycan-associatedprotein as defined by Lugtenberg et al. (21), anda similar finding has been reported by Verstreateet al. (42) for their group 2 proteins. In thepresent report, we extend these findings anddescribe a method for the isolation of the matrixprotein(s) of B. abortlus in a nondenatured state.A partial characterization of the 38K protein isalso presented.

MATERIALS AND METHODS

Bacterial cultures and cell envelope preparation. Thecharacteristics of B. abo)rtlus rough (R) strain 45/20 and

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B. ABORTUS MATRIX PROTEIN 395

smooth (S) strain 1119-3 and methods of preparing cellenvelopes have been described previously (13. 25).Briefly, logarithmic-phase cells were harvested bycentrifugation from cultures in Trypticase soy broth(BBL Microbiology Systems. Cockeysville. Md.) dial-ysate in flasks on a rotatory shaker. After beingwashed with saline, the cells were suspended in 10 mMHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesul-fonic acid) (pH 7.5), DNase and RNase (100 pLg/mleach) were added, and the cells were broken in acolloid mill (Mini-Mill. Greerco Corp.. Hudson.N.H.). Cell envelopes were recovered by ultracentrif-ugation and stored at -20°C in HEPES. E. coli K-121485 F+ cell envelopes were prepared by disruption ina French pressure cell as described by Diedrich et al.(5).

Preparation of Rosenbusch envelopes and extractionof matrix proteins. Peptidoglycan sacculi with theirassociated and covalently linked proteins (Rosenbuschenvelopes) were prepared by the SDS differential heatextraction method of Rosenbusch (36). Briefly. cellenvelopes were suspended in 10 mM Tris-hydrochlo-ride (pH 7.5)-2% SDS-0.7% 2-,B-mercaptoethanol-10% glycerol (SDS-fM-buffer) at 5 mg of cell envelopeprotein per ml, incubated at 50'C for 30 min. and thencentrifuged at 100.000 x g for 1 h. Where indicated.the pellet was reextracted under identical conditions.

Matrix proteins were extracted by three differentmethods. First. Rosenbusch envelopes were resus-pended in SDS-3M buffer and extracted at 100(C for Ito 2 min (36). After centrifugation at 100,000 x g for 1h. supernatant fluids containing the matrix proteinswere collected and stored at 4°C. Second, Rosenbuschenvelopes were extracted in SDS-,BM buffer contain-ing 0.5 M NaCI at 37°C for 30 min. and matrix proteinswere recovered after centrifugation as describedabove (30). Third. similar extractions were performedin SDS-[M buffer containing 50 mM MgCI.. Whennecessary. proteins were concentrated by precipita-tion with acetone and either redissolved in SDS-3Mbuffer or dialyzed extensively, first against 20()^X ace-tone in water and then against distilled water. andstored at -20'C.

Electron microscopy. Rosenbusch envelopes werenegatively stained with 1% neutral phosphotungsticacid and examined with a Hitachi HU 11 E electronmicroscope.

Gel filtration chromatography. Gel filtration wasperformed in Sephacryl S-300 (Pharmacia Fine Chemi-cals. Piscataway. N.J.) in columns (2.6 by 85 cm). Thebuffer systems used were 1) mM Tris-hydrochloride(pH 7.5)-0.25% SDS (Bio-Rad Laboratories. Rich-mond. Calif.)-5 mM EDTA-0.05% NaN, (SDS-EDTAbuffer) and 10 mM Tris-hydrochloride (pH 7.5)-0.25%SDS-l00 mM NaCI-0.05% NaN3 (SDS-NaCI buffer).The s.ame buffer systems with SDS replaced by Zwit-tergent 316 (Calbiochem. La Jolla. Calif.) were alsoused. Columns were calibrated with bovine serumalbumin, egg albumin. and lysozyme (Sigma ChemicalCo., St. Louis, Mo.). Stokes radii for these markers inSDS are 7.8, 5.8, and 2.75 nm. respectively (40).SDS-PAGE. SDS-PAGE was performed by the

method of Laemmli (18). Gels were stained for pro-teins with Coomassie blue (9) and for carbohydrateswith Schiff reagent by the method of Zacharius andZell (46). MW standards were phosphorylase B (94K).bovine serum albumin (67K). aldolase (40K). carbonic

anhydrase (29K). hemoglobin (4 x 16K) and cyto-chromec (12K).

Trichloroacetic acid-extracted, phenol-extracted.and purified phenol-extracted lipopolysaccharides(LPS) were prepared from B. thortuts 1119-3 as de-scribed previously (2. 24) and analyzed by SDS-PAGE. LPS-gels were stained for proteins by thealkaline silver method (32).

Preparative SDS-PAGE. Preparative SDS-PAGEwas performed as described by Chai and Foulds (3).Purified proteins were precipitated with trichloroace-tic acid at 4°C. dialyzed extensively against 20%acetone and water. and lyophilized.

Limited proteolysis in SDS-PAGE. Matrix proteins ofB. achor-tius 45/20 and Escherichia coli were first puri-fied by preparative SDS-PAGE. Alternatively, viablecells of B. abortus 45/20 and 1119-3 were extrinsicallylabeled by the [1251]lactoperoxidase catalysis method(22) and extracted with SDS-3M buffer at 100°C. AfterSDS-PAGE of these extracts, the gels were stainedwith Coomassie blue, and the selected proteins werecut from the gel. Limited proteolysis was carried outwith Staphx'lococcius aiureus V-8 protease (Miles Lab-oratories, Inc., Elkhart, Ind.) during SDS-PAGE (4.7). and the gels were stained with Coomassie blue orautoradiographed on Kodak XAR-5 X-ray film.

Analytical methods. 2-Keto-3-deoxyoctulosonic acid(KDO) was measured by the thiobarbituric acid meth-od corrected for interference by 2-deoxy sugars (25.43). Protein was determined by the method of Lowryet al. (20). When KDO and proteins were measuredwith material that otherwise would be dissolved orextracted in SDS-fM buffer. 2-,B-mercapthoethanoland glycerol were left out of the buffer. For fatty aciddetermination. 1 to 2 mg of purified protein washydrolyzed with 2 N HCI as described by Kates (16).and the methyl esters were analyzed in a Varian 3700gas-liquid chromatography apparatus equipped with aCDS 11I integrator with known amounts of archidateas internal standards. Fatty acid standards were ob-tained from Supelco. Inc., Bellefontaine, Pa.. andSigma.

Antisera. Antisera to cell envelopes of B. abortuts1119-3. B. abortuts 45/20. and E. coli were raised inNew Zealand white rabbits as described by Smyth etal. (38). Antibodies to matrix proteins were producedin outbred ICR mice. Protein bands cut from SDS-polyacrylamide gels after a brief staining were homog-enized in 0.15 M NaCl and administered intraperitone-ally in Freund complete adjuvant. at 5 fig/ml in 0.2 mlper animall. weekly for 4 weeks. Four days after thelast injection. ascites was induced with sarcoma 180cells. and ascitic fluid was collected (37).

Purification of rabbit IgG. The immunoglobulin G(IgG) fractions of rabbit antisera were purified bychromatography on protein A-Sepharose 4B (35). dia-lyzed against 20 mM phosphate-buffered saline (pH7.2)-0.05% NaN, and concentrated by ultrafiltrationon an Amicon OM 20 membrane.

Immunoelectrophoresis. Immunoelectrophoresis Walscarried out in 0.04 M sodium barbital-hydrochloridebuffer (pH 8.6) containing 1% Triton X-100 (EastmanKodak, Rochester, N.Y.; scintillation grade) in bothreservoirs and gels (1% agarose. Sigma type 1) for 60min at 5 to 10 V/cm with running tap water as thecoolant.ELISA. B. abor-tius S LPS was attached to polysty-

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396 MORIYON AND BERMAN

FIG. 1. Electron micrographs of B. abortus cell envelopes (A) and E. coli K-12 cell envelopes (B) extractedwith SDS at 55°C. Bar, 150 nm.

rene microtiter plates by overnight incubation of a 1,ug/ml solution in 0.06 M carbonate buffer (pH 9.6) at37°C. Rabbit anti-mouse IgG (Miles) was conjugated tohorseradish peroxidase (Sigma type VI) (31). Antigen-antibody reactions were allowed to proceed at roomtemperature overnight, and the enzyme-linked im-munosorbent assay (ELISA) was performed as de-scribed previously (19).

RESULTSDifferential heat extraction of matrix proteins.

To determine the presence in B. aborttis of a

matrix protein(s), we first used sequential SDS-heat extraction (36). Electron micrographs ofcell envelopes extracted with SDS-rM buffer at50°C showed the arrangement of E. coli matrixproteins on the peptidoglycan and a similarimage for B. abortuis (Fig. 1). Upon reextractionat 100°C in SDS-1M buffer, both the E. (oli andthe B. abortus matrix proteins were removedfrom the peptidoglycan, and a conspicuous pro-tein with a MW of about 38K was detected bySDS-PAGE in the extracts, along with smaller

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B. ABORTUS MATRIX PROTEIN 397

1 2 3

94k-k

67k

40k..-

29k -

A1 2 3 4

94ki

40k 9 W

29k

B2 4

94k

67k

# 40k

29k

16k-.12k-

FIG. 2. SDS-PAGE analysis of matrix proteinsprepared by the differential SDS-heat extraction meth-od of Rosenbusch (36). Lanes: 1. B. abortuis 45/2)0: 2

B. aborutis 1119-3; 3. E. coli K-12.

amounts of several other proteins of similar MW(Fig. 2). As with E. (coli. B. abor-tius matrixproteins were released from the peptidoglycanby SDS at temperatures above 60°C. However.in contrast to E. coli, when Rosenbusch enve-lopes of B. abortlis were prepared, it was neces-

sary to repeat the extraction at 50°C to obtainlow levels of background in SDS-PAGE. Ananalysis of the supernatants of successive ex-

tractions with SDS-,M buffer at 50°C confirmedthat background levels of both protein and KDO

1 2 3 4

-

FIG. 3. SDS-PAGE analysis of peptides obtainedby limited proteolysis of matrix proteins. Proteinswere prepared as described in the text, and proteolysiswas carried out during stacking with 2 p.g of S. alurelusV-8 protease. Gels were stained with Coomassie blue(lanes 1 and 2) or autoradiographed (lanes 3 and 4).Lanes: 1. E. coli K-12: 2. B. abortlus 45/20: 3. B.aboru-tis 45/20; 4. B. abortuis 1119-3. Arrows. E. coliand B. abortuis proteolytic fragments of similar MW.

FIG. 4. SDS-PAGE analysis without urea (A) or inthe presence of 5 M urea (B) of matrix proteinsextracted from Rosenbusch envelopes with 50 mMMgCI. in SDS-4M buffer at 37°C. Lanes: 1. E. coli K-12 incubated at 37°C; 2. E. coli K-12 incubated at100°C; 3. B. abortius 45/20 incubated at 37°C; 4. B.aborti s 45/20 incubated at 100°C.

were reached more easily for E. coli than for B.aborutis (not shown). Even after repeated ex-traction at 50'C, B. aborttis matrix protein prep-arations also contained an 88K protein (Fig. 2).A protein of similar MW was removed from E.coli cell envelopes during the first extractionwith SDS at 50°C.The apparent MW of B. aborttis 45/20 matrix

proteins in SDS-PAGE was somewhat less thanthat of B. abor-tius 1119-3 or E. (coli matrixproteins (Fig. 2). Peptide profiles after limitedproteolysis of matrix proteins from both S and RB. aborttis were almost identical and were dis-tinct from that of E. coli matrix protein (Fig. 3).The accessibility, in this experiment, of matrixproteins of B. abortius to extrinsic labeling by the[1251]lactoperoxidase catalysis method indicatedtheir exposure on the surface of both R and Sstrains.

Extraction of matrix proteins by NaCI andMgCI2. Although E. coli matrix proteins wereextracted in a nondenaturated state from Rosen-busch envelopes by 0.5 M NaCl in SDS-rMbuffer at 37°C (30), B. aborutis matrix proteinswere not. However, MgCI. in SDS-,BM buffer at37°C extracted the matrix proteins, as well asLPS and smaller amounts of other proteins.from Rosenbusch envelopes of both B. (abortutsand E. (oli. The best yields in a single extraction(60% of the matrix protein extracted at 100°C)were obtained at MgCI. concentrations of 50mM or greater for B. abo r-tis and 100 mM for E.coli. When SDS was replaced by Zwittergent316, 10 to 20 mM MgClI was enough to extractthe matrix proteins from Rosenbusch envelopesof both bacteria.

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398 MORIYON AND BERMAN

Characterization of B. abortus matrix proteins.B. abhortius matrix proteins extracted by MgCl,-SDS-4M buffer at 37°C were precipitated withacetone and incubated in SDS-rM buffer at37°C. When these preparations were examinedby SDS-PAGE, the 38K protein remained at thetop of the tracking gel (MW, >94K) (Fig. 4A).After dialysis against 20% acetone and water,retained SDS was about 0.001% of the total dryweight as determined by gas-liquid chromatogra-phy. After incubation in SDS-3M buffer at tem-peratures above 60°C, the protein had an appar-ent MW of 38K (Fig. 4A). After acetone-waterdialysis of this preparation, SDS accounted for 2to 3% of the total dry weight. Although both theMW shift and reversible SDS binding of thematrix proteins incubated at 37°C were similarfor E. coli (30, 36), only traces of SDS weredetected in matrix proteins of E. coli incubatedin SDS-,M buffer above 60°C after acetone-water dialysis. Furthermore, although the mobil-ity of E. coli matrix proteins denatured in SDS at1O0TC decreased in SDS-5 M urea gels withrespect to that in SDS gels, B. abortis matrixproteins had the same mobility in both gels (Fig.4B).LPS in matrix protein preparations. As stated

above, KDO was released during the prepara-tion of Rosenbusch envelopes until it reached abackground level. Upon extraction with MgCl2-SDS-3M buffer at 37°C, or at 100°C in SDS,additional KDO was released (data not shown).An immunoelectrophoretic analysis with antise-ra to whole cell envelopes revealed a majoranodally migrating component in matrix proteinpreparations dissolved in SDS (Fig. SA). Asecond precipitate line, formed by a diffusesmear from the first one, was observed only inpreparations from S B. abor-tims. An SDS-PAGEanalysis of the two precipitin lines showed thatthe 38K protein was present in the anodal pre-cipitate but not in the second precipitate (Fig.SB). This second precipitate was identified as SLPS by immunoelectrophoresis (Fig. SA). Thereaction of identity between these two precipitinlines suggests that interactions of LPS with theprotein persist during electrophoresis and thatthe LPS exists in both loosely and more firmlyprotein-bound states.SDS-PAGE of matrix proteins of the R strain

extracted with MgCl,-SDS-PM buffer showedthat carbohydrate-staining material also existedin free and protein-associated forms (data notshown). This association existed with proteinsincubated at either 37 or 100°C. Evidence thatthe carbohydrate-staining material represented,at least in part, LPS associated with the proteinwas obtained by detection, by ELISA, of anti-body to S LPS in ascitic fluids of mice immu-nized with purified matrix protein. The possibili-

ty that small amounts of matrix proteincontaminating the S LPS used as the antigen inELISA could be responsible for these results(10) was tested in two different ways. First,although preparations of trichloroacetic acid-extracted and crude phenol-extracted Briuella SLPS both contained a 38K-MW protein, demon-strable in silver-stained SDS gels, this proteinwas absent from purified phenol-extracted SLPS (not shown). Second. similar titers wereobtained regardless of whether ELISA was donewith the crude or the purified phenol-extractedLPS.When MgCl2-SDS-PM matrix protein extracts

were chromatographed in Sephacryl S-300 inSDS-EDTA buffer, most of the matrix protein ofB. aibortius was recovered in the void volume,with a small fraction of the same protein elutingin a second peak (Fig. 6A). A third peak con-tained the 30K protein, and LPS was partitionedamong the three peaks. When the first andsecond peaks were pooled and rechromato-graphed in SDS-NaCl buffer, most of the proteineluted in the second peak, and most of the LPSeluted after the protein (Fig. 6B). The B. abortisprotein peak eluted at the same position as thenondenatured E. coli matrix protein (5.3 nm).Although Zwittergents with alkyl chain lengthsgreater than 10 are very effective in dispersingLPS (25), when Zwittergent 316 was substitutedfor SDS, the protein standards, matrix protein,and LPS were all eluted in the void volumeregardless of the ionic strength of the buffer (notshown).

A B

FIG. 5. (A) Immunoelectrophoretic analysis of B.abortius 1119-3 S LPS and matrix proteins of theindicated strains of B. abortius (wells): troughs con-tained rabbit IgG against whole crude cell envelopes ofthe indicated strains. (B) SDS-PAGE of the anodicprecipitin line of B. abotutis 1119-3 (lane 1) and the B.abortus 1119-3 precipitin line closer to the well (lane2). The smears in SDS-PAGE were due to the pres-ence of Triton X-100 and represent the heavy and lightchains of the IgG. The matrix protein in lane 1 isindicated by the arrow.

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B. ABORTUS MATRIX PROTEIN 399

Vo 71A )nA :. A

I * I I Pg LPS ml

B O 200

;.0 SDS NaCI !io

0.50 '. ~~~50

K 'o - 4- ---V 150 200 21 0

KDO measurements showed that LPS re-mained associated with the protein after repeat-ed gel filtration in SDS-NaCl buffer (1.6 pg ofKDO per mg of protein). In an attempt toremove this LPS, the protein was denatured byboiling in SDS at 100°C and purified further bypreparative SDS-PAGE, and its fatty acid con-tent was then determined by gas-liquid chroma-tography (Table 1). Although these preparationscontained significant amounts of fatty acids thatare also present in the LPS, the relative propor-tions of the acids differed from those reported

TABLE 1. Fatty acid composition of B. taborustti SLPS and B. abortuis matrix protein purified by

sequential gel filtration in SDS-EDTA and SDS-NaCIbuffers followed by preparative SDS-PAGE'

% of total fatty acid in:Fatty acidh

LPS Prepn I Prepn II

A16:016:1B18:018:1CDEF

5.476.40.4

12.329.96.34.6

6.9 8.65.4 21.5

5.63.8

1.3 2.17.3 5.6

FIG. 6. Sephacryl S-300 gel filtration of matrixproteins. (A) B. abolrttis 45/20 matrix proteins extract-ed with 50 mM MgCI. in SDS-3M buffer, equilibrated,and chromatographed in SDS-EDTA buffer. (B) V()and second peaks of (A) pooled. equilibrated, andchromatographed in SDS-NaCl buffer. Symbols: C.KDO (LPS); 0. protein. OD 280. Optical density at280 nm.

for the LPS or cell envelopes of B. abortits (1,24). E. coli matrix protein preparations purifiedand analyzed by the same methods containedtraces of ,B-hydroxymyristic acid and seven tonine times less total fatty acid per milligram ofprotein than that in preparations from B. (abor-tils.

DISCUSSIONOur results show that cell envelopes of B.

abortiis contained a protein similar to the matrixprotein of other gram-negative bacteria (14. 21,23, 36). The 38K protein was not extracted bySDS at temperatures below 60°C and remainedclosely associated with the peptidoglycan. Attemperatures above 60°C, the protein underwentdenaturation, was extracted from the peptido-glycan, exhibited its 38K MW in SDS-PAGE.and bound SDS strongly. Matrix proteins pres-ent in S and R strains had slightly differentapparent MWs. Since as many as three proteinswith MWs clustered around 38K were observed,it is possible that the two strains studied mightexpress different major proteins. Alternatively,tightly bound LPS, along with differences in thecarbohydrate content of S and R LPS (24). couldexplain different mobilities for an otherwiseidentical protein. In view of the presence of sucha tightly protein-bound LPS and the identicalproteolytic patterns given by both proteins, wefavor the second alternative.

In contrast to E. coli matrix proteins, B.abortius matrix proteins were not extracted byNaCl in SDS-PM buffer at 37°C. Both E. coli and

" Results obtained with two different matrix proteinpreparations (I and II) are presented. The total fattyacid content was 15 p.g per mg of protein for prepara-tion I and 18 pg per mg of protein for preparation 11.

h Letters represent unknown fatty acids.

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400 MORIYON AND BERMAN

B. aborttis matrix proteins were extracted byMgCl2 in SDS-4M buffer at 37°C. The B. abortis45/20 matrix proteins prepared by extractionwith MgCl2 in SDS-rM buffer were in a nonde-natured state, as shown by (i) their ability toreact with antibodies produced against cell enve-lopes, (ii) the fact that, in contrast to the proteinsdenatured by SDS at 100°C, SDS could becompletely removed, and (iii) their identity withnondenatured (31) E. coli matrix proteins withrespect to mobility in SDS-PAGE and behaviorduring gel filtration in SDS. Since nondenaturedE. coli matrix protein has a Stokes radius of 5.3nm in SDS (45), this should also be valid for B.abortus matrix protein (40). A Stokes radius of5.3 nm and association with the peptidoglycanimply that the protein should span the outermembrane, which is in agreement with the ac-cessibility of the 38K protein to extrinsic label-ing.LPS contaminating the matrix protein prepa-

rations was in both free and protein-bound statesin the presence of detergents. Most contaminantproteins and LPS were removed by sequentialgel filtration in SDS-EDTA and SDS-NaCl buff-ers. The differences observed between the pro-tein distributions in SDS-EDTA and SDS-NaClbuffers were presumably due to different statesof aggregation of the protein itself (I. Moriyonand D. T. Berman, unpublished data), and simi-lar observations have been made for enterobac-terial matrix proteins (28, 41). The remainingLPS was tightly bound and could not be re-moved by preparative SDS-PAGE. Although B.aborilis LPS lacks fatty acids that, like 3-hy-droxymyristic acid of enterobacterial LPS, canbe used unequivocally as LPS markers, most ofits fatty acids consist of palmitic, stearic, andoleic acids (1, 24). The relative proportions ofthese fatty acids associated with Brucella matrixprotein were different from those of either theLPS or the cell envelopes (1, 24), and theyprobably represent a mixture of lipids from bothorigins.We have previously interpreted the resistance

of the B. abortus cell envelope to nonionicdetergents and EDTA (25) as indicating that it ismore hydrophobic than that of E. coli. Some ofthe differences between B. abortus and E. colimatrix proteini may also be understood in thatcontext. First, for B. abortus, but not for E. coli,SDS extraction at 50°C had to be repeated toreduce the amounts of contaminating LPS andprotein. This could be the result of strongerhydrophobic associations among the envelopecomponents. Second, urea reduces the interac-tion of SDS with E. coli OmpC matrix protein(45), and this could account for its apparent MWshift in urea gels (34). It is possible that theinteraction between SDS and B. abortius matrix

protein is stronger and, therefore, that it wouldnot be affected to the same extent by the chao-tropic-like action of urea (11). This interpreta-tion is consistent with the observation that B.aborutits matrix protein retained substantiallymore SDS than E. (oli matrix protein afteracetone-water dialysis. Finally, B. abor-tius ma-trix protein prepared by SDS-PAGE retained asignificantly higher amount of tightly bound lipidthan E. coli matrix protein prepared in a similarfashion.

Recently, Verstreate et al. (42) have identifiedand partially characterized three groups of pro-teins from outer membranes of B. abortuis. Itseems likely that their group 2 protein clusterand the 38K protein identified by us are identi-cal. They found that LPS copurified with each oftheir three groups of proteins in ion-exchange,hydroxyapatite, and gel filtration chromatogra-phy, all performed in Zwittergent 314. They alsoreported that proteins belonging to differentgroups copurified in ion-exchange chromatogra-phy, although the patterns were not consistent.Our results suggest that the high micellar weightof the Zwittergents could be responsible, at leastin part, for those associations. Similar problemshave been encountered by others (12) in thepurification of E. coli outer membrane proteinsin the presence of Triton X-100.The extraction and purification procedures

described in this work provide an alternativemethod to those described by Dubray and Be-zard (8) and Verstreate et al. (42) for obtainingB. abortius matrix proteins in a nondenaturedstate for investigation of their immunologicaland biological properties. It will be necessary insuch investigations to take into account effectsthat could be attributable to the tightly boundLPS (10, 15, 17).

ACKNOWLEDGMENTS

We are grateful to B. D. Whitaker for performing the gas-liquid chromatography analysis and to M. K. Hayes and B.Alonso-Urmeneta for excellent technical assistance.

This investigation was supported by the College of Agricul-tural and Life Sciences, University of Wisconsin, by Coopera-tive Agreements with the U.S. Department of AgricultureAnimal and Plant Health Inspection Service and the Agricul-tural Research Service, and by fellowship support for I.M.from the U.S.A.-Spanish Joint Committee on Scientific andTechnical Cooperation.

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2. Boivin, A., and L. Mesrobeanu. 1935. Recherches sur lesantigenes somatiques et sur les endotoxines des bacteries.1. Considerations generales et expose des techniquesutilisees. Rev. Immunol. 1:553-569.

3. Chai, T., and J. Foulds. 1977. Purification of protein A, anouter membrane component missing in Escherichio oliK-12 OmpA mutants. Biochim. Biophys. Acta 493:210-215.

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4. Cleveland, D. W., S. G. Fischer, NI. W. Kirshener, andU. K. Laemmli. 1977. Peptide mapping by limited proteol-vsis in sodium dodecyl sulfate and analysis by gel electro-phoresis. J. Biol. Chem. 252:1102-1106.

5. D)iedrich, D. L., A. 0. Summers, and C. L. Schnaitman.1977. OLIter membrane proteins of Escherichia co/i. V.Evidence that protein 1 and bacteriophage-directed pro-tein 2' are different polypeptides. J. Bacteriol. 131:598-607.

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