JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 1-6 Vol. 164, No. 10021-9193/85/100001-06$02.00/0Copyright © 1985, American Society for Microbiology

Antigenic Mosaic of Methanosarcinaceae: Partial Characterizationof Methanosarcina barkeri 227 Surface Antigens by

Monoclonal AntibodiesJUAN C. GARBERI, ALBERTO J. L. MACARIO,* AND EVERLY CONWAY DE MACARIO

Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201

Received 28 May 1985/Accepted 26 June 1985

Hybridomas were constructed with spleen cells from mice immunized against Methanosarcina barkeri 227.The reaction with the resulting monoclonal antibodies identified two antigenic determinants. Determinant 8Ais present in M. barkeri 227, where it is accessible to antibody on whole bacterial cells. 8A is undetectable in(or absent from) M. barkeri R1M3, an immunologically closely related strain. Determinant 8C is present inboth strains, but with M. barkeri 227 it is found only in extracts and cannot be demonstrated in whole cells. Ittherefore appears to be hidden. A soluble form of antigen 8A (antigen 227) was obtained treating whole M.barkeri 227 cells with absolute methanol. This antigen was further purified by affinity chromatography withantibody 8A. Chemical and immunochemical analyses of these preparations showed that antigen 227 is ahigh-molecular-weight (4 x 105) structure composed mainly of one carbohydrate, glucose, and small amountsof amino acids. Its solubility properties suggest that this molecule is associated with a lipid moiety.

Because members of the family Methanosarcinaceae (2)are a distinct group of microorganisms, closely relatedamong themselves (22) yet distant from other Archae-bacteria, they constitute an interesting target for studies ofprocaryote evolution. Methanosarcinae are also importantfor biotechnology; they play a major role in anaerobicfermentation and are potentially useful for the elimination oforganic wastes (9, 17, 18, 29-31).Although much remains to be learned about methano-

sarcinae, studies of their physiology and biochemistry (2, 10)strongly suggest an early evolutionary separation from othermethanogenic bacteria. This separation probably explainswhy their surface architecture developed differently fromthat of other methanogens and procaryotes. The cell wall,for example, does not contain the usual peptidoglycan;rather, it is a heteropolysaccharide, composed of glucuronicacid and galactosamine (13). Their lipid composition is alsodifferent from that of other methanogens, consisting mainlyof C-20 diphytanyl diethers of glycerol (23).The three-dimensional structure of the cell surface of

methanosarcinae has not yet been established, mainly be-cause of the lack of specific probes for individual compo-nents. Using whole cells as antigen should provide an arrayof antibodies to undertake this task. However, only mono-clonal antibodies would have the necessary specificity.For this purpose we have generated a panel of monoclonal

antibodies against Methanosarcina barkeri (C. G. Schnel-len, dissertation, Technical University of Delft de Maastad,Rotterdam, 1947) strain 227 (17); three of these antibodieswere well characterized. With these probes we have begunto elucidate the structure of the methanosarcinae envelope.Here we report two antigen-bearing structures located atdifferent levels of the cell surface. One of these antigens hasbeen purified by affinity chromatography and partially char-acterized chemically.

* Corresponding author.

MATERIALS AND METHODS

Bacteria and antigen preparations. M. barkeri strains 227(17) and R1M3 (6) were obtained from M. J. Wolin and hiscollaborators at the Wadsworth Center and were prepared forimmunologic study as previously described (6). Bacterialhomogenates were prepared by mechanical shearing of M.barkeri 227 cells in an equal volume of distilled water with1-mm-diameter glass beads (15). The cell homogenate,separated from the beads by pipetting, was utilized as anantigen source without further treatment. It was stored at-20°C. To obtain soluble antigen, 40 mg of M. barkeri 227cells was washed three times with phosphate-buffered saline(PBS) and dispensed into two tubes with 1 ml of PBS orabsolute methanol. The tubes were incubated for 4 h at 25°Cand then for 16 h at 4°C in an end-over-end rotator. They werethen centrifuged at 1,500 x g for 30 min, and the uppertwo-thirds of each supernatant was decanted and furthercentrifuged at 15,000 x g for 10 min. The upper two-thirds ofeach supernatant was saved. A 0.5-ml sample of each wasassayed for antigenicity, and the rest was stored at -20°C.(The protocol for bulk purification of antigen 227 is describedin detail under Results.)Monoclonal antibodies. Monoclonal antibodies 8A, 8B,

and 8C, anti-M. barkeri 227, were prepared, characterized,and calibrated by standard procedures (19). Large quantitiesof these antibodies were obtained by cultivating the hybridcell lines in 15-ml flasks and collecting the used medium. Forpurification, 50 ml of this medium was applied to a 0.9- by10-cm column of protein A-Sepharose 4B (Pharmacia FineChemicals) (8), and immunoglobulins were eluted by sequen-tial washings with buffers of decreasing pH. The pH 6.0fraction was further purified on a 0.9- by 15-cm column ofDEAE Affi Gel Blue (Bio-Rad Laboratories) with a 0.0 to 0.5M NaCl gradient in 0.05 M Tris buffer, pH 7.4 (Bio-Radbulletin 1069, 1982). Titers of antibody were determined withthe slide immunoenzymatic assay (4, 5). For each antibody aworking dilution was chosen, i.e., the one that generated a

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TABLE 1. Characteristics of the monoclonal antibodies

ReactionCell line Antibody IgG IgG Working with straind:(mg/ml)a subclassb dilutionc

227 R1M3

L8A 8A 0.125 IgGl 64 4+ -L8B 8B 0.125 IgGl 16 2 + -L8C 8C 0.370 IgGl 600 - 4+

Myelomae None 0 NAf NA -

a Determined by radial immunodiffussion.b Determined by protein A-Sepharose chromatography and slide im-

munoenzymatic assay (4).c Determined as previously described (5).d Determined by semiquantitative slide immunoenzymatic assay (6).eSp2/0-Agl4, a nonsecretor mouse myeloma cell line used in our fusion

experiments.f NA, Not applicable.

preestablished optical density at 450 nm 30 min after theaddition of enzyme substrate.

Inhibition-blocking experiments. The molecular specifici-ties of the monoclonal antibodies were determined by inhi-bition-blocking experiments (5). The antigens were unfixedand formalinized whole cells of M. barkeri 227 and R1M3,PBS extracts of these cells, and the following 15 compoundsof known structure: D-glucose, D-rhamnose, D-mannose,D-galactose, and D-galacturonic acid from Calbiochem-Behring; D-glucuronic acid, D(+)-glucosamine hydro-chloride, D(+)-galactosamine hydrochloride, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, L-fucose,L-omithine, L-methionine, and L-cystine from Sigma Chem-ical Co.; and sucrose from Eastman Chemical Products, Inc.

Affinity chromatography. Purified monoclonal antibody 8Awas coupled to CnBr-activated Sepharose 4B (Pharmacia)by the method recommended by its manufacturer (1). Sam-ples (0.5 to 1 ml) in PBS (pH 7.4) were applied on the 0.9- by3-cm column and allowed to equilibrate for 30 min.Nonadsorbed compounds were washed off with 5 columnvolumes of PBS. One column volume of distilled water wasthen applied and allowed to interact with the antigen-antibody complexes for 30 min. Bound antigens were elutedwith 2 more column volumes of distilled water, followed by2 column volumes of 5% NH40H.

Antigen measurements. Antigenicity was measured bydirect-binding slide immunoenzymatic assay with monoclo-nal antibodies 8A and 8B, (specific for strain 227 antigen) and8C (specific for strain R1M3 antigen). Absorbance at 450 nmwas read 30 min after the enzyme substrate was added.

Chloroform-methanol extraction. Samples of a methanolextract of antigen 227 were dialyzed against PBS. Theconcentration of NaCl was adjusted to 0.2 M, and an equalvolume of chloroform-methanol (2:1, vol/vol) was added andmixed. The tubes were left standing at 25°C for 15 min andthen centrifuged at 1,500 x g at 4°C for 15 min. The organicand aqueous phases were separated, evaporated, and sus-pended in absolute methanol. Samples were tested forantigenicity.

Analytic methods. Neutral sugars were determined byhydrolysis in 2 N HCI at 1000C for 2.5 h, followed byautomated borate-ion-exchange chromatography (26) withfluorescence detection (11). Hexosamines were analyzedwith an amino acid analyzer after hydrolysis in 2 N HCl for6 h (20). Amino acids were determined after hydrolysis in 6N HCl at 110°C for 24 h. For all analyses the tubes wereevacuated four times and flushed with N2. Column effluents

were monitored for peptides by reacting samples withfluorescamine (25). Total neutral carbohydrates were mea-sured by the phenol-sulfuric acid method (7).Paper chromatography. Descendent chromatography pa-

per (Whatman no. 1) was used with n-butanol-pyridine-acetic acid-water (420:280:21:210, voUvol) as thesolvent. Chromatograms were developed for reducing sugarswith silver nitrate reagent (24).

Thin-layer chromatography. Silica gel G chromatographywas performed on 300-,um, precoated plates (Analtech).Four sets of six plates each were exposed to the followingsolvents: n-butanol-pyridine-water (65:34:17, voUvol), chlo-roform-methanol-water (65:34:8, vol/vol), methanol-water(70:30, vol/vol), and 1-propanol-acetic acid-water (8:1:4).

After chromatography the plates in each set were treatedas follows: plate 1, iodine vapors for 1 h; plate 2, 50%sulfuric acid in ethanol and heat (300°C), 2 h; plate 3, 5%phenol plus 3% sulfuric acid in water and heat (110°C), 30min; plate 4, 5% ninhydrin in ethanol and heat (80°C), 30min; plate 5, bromothymol (0.04%) in 0.01 N NaOH; andplate 6, 2', 7'-dichlorofluorescein (0.2%) in ethanol (96%).Chromatograms were examined under UV light.

RESULTSSpecificity spectrum of hybridoma products. In one exper-

iment, 220 microcultures were set up and screened forantibody reactivity. Of these, one group reacted exclusivelywith the immunizing strain, M. barkeri 227. Another groupreacted with M. barkeri 227 and a heterologous but immu-nologically related strain, R1M3 (6, 16). A third groupreacted exclusively with M. barkeri R1M3. The cell linesderived from the second group were highly unstable andwere not considered for these studies.Three cell lines were cloned, subcloned, and kept in

culture through successive passages until 1 liter of theirsupernatants was collected. Two produced antibodies spe-cifically against M. barkeri 227; the third produced antibod-ies specifically against M. barkeri R1M3 (Table 1).

0.5r 8A

E 0.3a

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< 0.3-

227 RIM3 '- 227--'WHOLE CELLS SOLUBLE PREPAI

LR1M3J 227

RATIONS CELLHOMOGENATE

ANTIGEN PREPARATIONSFIG. 1. Reaction of monoclonal antibodies 8A and 8C with

various antigens preparations. Symbols for the soluble preparations:I2, PBS preparation stored for 2 weeks at 4°C; M, used culturemedium; E, methanol extract.

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TABLE 2. Partial composition and antigenicity of methanol extract and purified antigen 227

AntigenicityAntigen prepn Amino acid (nmollml)a Carbohydrate (nmol/ml)b CHO/aa ratioc Specifice

Total (U/W.)" pcfc(mU/nmol of aa) (mU/nmol of CHO)

Methanol extract 90 61 0.68 0.4 4.4 6.6Purified antigen 227 38 305 8.02 12.0 315.8 39.3

a Free amino acids were determined by the fluorescamine assay (25).b Neutral sugars were determined by phenol-sulfuric acid assay (7).c Moles of carbohydrate per mole of amino acid.d One unit of antigenic activity was arbitrarily established as the amount of antigen 227 in 10 p.1 of a solution which gives an absorbancy at 450 nm of 0.20 when

antigenicity is measured by direct-binding assay with a standard mnonoclonal antibody 8A probe, using the slide immunoenzymatic assay at 23aC and readingabsorbance 30 min after the addition of enzyme substrate.

e Specific antigenic activity: milliunits of antigen 227 in a given volume per mole of amino acids (aa) or carbohydrate (CHO) in the same volume.

Each antibody was purified to homogeneity, and itsmonoclonality was confirmed by isoelectrofocusing, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, and spe-cific absorption with the homologous bacteria. All threewere immunoglobulin Gl (IgGl). Titers of the antibodieswere then determined by slide immunoenzymatic assay withthe homologous bacteria so that they would be used at anequal level of activity in the following experiments (workingdilution). Antibody 8A had a higher titer than antibody 8B,even though both cell lines secreted the same amounts ofmouse IgGl (Table 1).

Inhibition blocking with compounds of known structure. Ofthe 15 compounds tested, only glucose caused an inhibition-blocking effect on antibody 8A (25 + 3.3%, mean + standarddeviation; n = 6). Glucose, however, did not inhibit mono-clonal 8B, which showed the same specificity as monoclonal8A when assayed with whole cells.

Presence in M. barkeri 227 of determinant recognized byantibody 8C. Although antibody 8C was prepared by usingformalinized whole cells of M. barkeri 227 as the im-munogen, it did not react with M. barkeri 227 whole cells,whether formalinized or unfixed (Fig. 1). This finding sug-

gested that the antigenic determinant for antibody 8C ismasked in whole cells, but becomes exposed when the cellsare injected into mice.To test this hypothesis, water-soluble extracts from the

envelopes of M. barkeri 227 and R1M3 were assayed withantibodies 8A and 8C. Antibody 8A reacted only with thesoluble preparations from strain 227, Whereas antibody 8Creacted with the soluble preparations from both strains (Fig.1). Identical results were obtained with homogenates ofwhole M. barkeri 227 cells. These observations were furtherevidence that the antigenic determinant for antibody 8C isnot exposed on the surface of intact M. barkeri 227.

Selective extraction of the structure recognized by antibody

TABLE 3. Carbohydrate composition of antigen 227

Methanol extractPurifiedCarbohydrate Prfe

%aCMolar antigen (%)ratiob

Rhamnose 9.6 0.3 NDCMannose 56.1 1.7 NDGlucose 34.0 1.0 100Glucosamine 0.3 <0.1 ND

a Percentage: (moles of each carbohydrate [e.g., rhamnose]/moles of totalcarbohydrates) x 100.

b Molar ratio to glucose: moles of each carbohydrate/moles of glucose.cND, Not detectable.

8A (antigen 227). Soluble antigen preparations were obtainedby treating the cells with organic solvents. Extraction withabsolute methanol proved to be the most effective. In only 24h 87% of the antigen 227 found in cell homogenates wasselectively extracted. Monoclonal 8C did not react with thispreparation, indicating that R1M3 antigen was not solubil-ized here (Fig. 1). Two subsequent extractions considerablyimproved the efficiency of extraction without contaminatingthe sample with antigen R1M3.

Chemical analysis and chromatography of antigen 227methanol extract. Preliminary chemical analysis of the meth-anol extract from M. barkeri 227 cells revealed that it is acomplex mixture which contains free-amino compounds andcarbohydrates (Table 2). Thin-layet chromatography on sil-ica gel G, developed with general reagents such as iodinevapors and phenol-sulfuric acid, showed at least five com-ponents with relative mobilities of Rf 0, 0.2, 0.35, 0.69, and0.84. In contrast, purified antigen 227 yielded a single definedspot at the origin.

Tests for lipids with general reagents such as bro-mothymol blue and 2',7'-dichlorofluorescein were negative.When the methanol extract was dialyzed against sodiumphosphate buffer (10 mM, pH 7.4) containing sodium chlo-ride (150 mM) and then extracted with a 2:1 mixture ofchloroform-methanol, 90 to 95% of the antigenic activity wasfound in the organic phase (data not shown)-a strong indi-

TABLE 4. Amino acid composition of antigen 227 preparations

Methanol extract Purified antigenAmino acid

Molar ratiob % Molar ratio

Asp 10.9 3.1 8.9 2.1Thr 4.4 1.3 4.6 1.1Ser 3.8 1.1 15.8 3.7Glu 49.9 14.3 15.4 3.6Pro NDC ND 3.7 0.8Gly 7.3 2.0 17.6 4.1Ala 5.0 1.4 8.2 1.9Val 3.7 1.0 4.2 1.0Met 1.9 0.5 ND NDIso 3.4 1.0 2.3 0.5Leu 3.5 1.0 4.4 1.0Tyr 0.6 0.1 ND NDPhe 0.8 0.2 1.9 0.5His ND ND 6.7 1.6Lys 3.6 1.0 4.2 1.0Arg 1.2 0.3 2.1 0.5

a Percentage: (moles of each amino acid/total moles) x 100.b Molar ratio to lysine: moles of each amino acid/moles of lysine.c ND, Not detectable.

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for 90ogm8,twnamn.ccotainng paksd (0).lsiAntigeni activityl(0)twastfoundamostl

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moalnsetand quifedantitiesno rhamfnoseg Aoosarcgfremn wihexoamieshinghydrolysates (2.5entrHtio atd gluta-fo,6h)shwetoltracenflcsaminsciiyreotdfrM (Tabler 3S(1).otherticarbohydratex foundmnthesempreprations.th

ftemethanolextract(Ti.2)soeohahg-ableshow-oedular-higher conentatio(fatofsglutamicgthota)gha repurtivedy) Eantien findinaiindgloodscagement-pwithite highpgutdsamatcaroncenration gnluta-matehtransamias-eaciigtyreported forigeMi barkeiviy MSe(14).

AnlyiclSepadx0-2column chromatography ofthipeksoedmethaenol extracty (ig. 2a)shfrctowaed both anhgh andraglow-molcularwegho 4x (Frcions9B) Throuigh 18landl20throght reispectively)Easaho peakicontained fluores-o

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ultrafiltered through an Amicon X300 filter (exclusion limit,3 x 105 daltons). The antigenic activity of these fractions wasfully retained by this membrane.

Antigen 227 was flanked by two major fluorescamine-positive peaks having molecular weights of >2 x 106 (frac-tions 9 through 13) and 4 x 104 (fractions 28 through 37).These peaks lacked antigenic activity (Fig. 2B). The high-molecular-weight peak was rich in neutral sugars, notablymannose, but comparatively poor in amino acids. The low-molecular-weight peak was rich in acidic amino acids, espe-cially glutamic acid, which constituted 26.3% of the total.The latter peak also had low concentrations of poly-saccharides made up of glucose, which accounted for 94.4%of the total sugar (data not shown).

Purification of antigen 227. The different steps involved inthe obtainment of purified antigen 227 are depicted in Fig. 3.

Affinity chromatography was chosen as the final step inthe purification because of the high specificity and easyavailability of purified monoclonal antibody 8A. By combin-ing a distilled water elution (3) with a mild alkaline elution(ammonium hydroxide, 5% in water) we were able to quan-titatively recover the antigen from the affinity column (Fig.4). The degree of purification achieved is reflected in theenhanced specific activity of the purified antigen (Table 2). Apaper chromatography system designed to separate carbo-hydrates and oligosaccharides showed a single spot withpurified antigen 227, which retained its antigenic activityafter elution with water. After acid hydrolysis (2 N HCI at100°C for 2.5 h), the purified antigen lost antigenicity andmigrated like glucose.

DISCUSSIONThree lines of evidence suggest that the cell surface of M.

barkeri 227 is a complex, multilayer envelope.(i) Although monoclonal antibodies 8A, 8B, and 8C were

raised against whole M. barkeri 227 cells, 8C does notrecognize whole cells as antigens. It reacts strongly, how-ever, with the cell homogenate (Fig. 1). The structure it doesrecognize appears, therefore, to be hidden deeper than thecell surface.

(ii) Both antigens (227 and the one defined by antibody 8C)are shed into the fluid phase of a cell suspension, i.e., theculture medium in which the bacteria are growing or the PBSin which they are stored (Fig. 1). However, antigen 227 isfound in much larger quantities than antigen R1M3 in thesepreparations, indicating for the former an easier accessibilityto the medium as well as less stringency in the bonds thathold this structure to the cell surface.

(iii) Monoclonal antibody 8C recognizes whole cells of M.barkeri R1M3, a closely related strain, implying that thisantigen is a cell surface constituent of R1M3. Analysis ofisolated cell wall fragments from M. barkeri 227 and immu-noelectron microscopy with monoclonal antibodies 8A and8C will help in establishing the locations of the two corre-sponding antigenic determinants in 227 cells.

Purified antigen 227 is richer in carbohydrate than themethanol extract. Glucose appears to be its only carbohy-drate component, although the methanol extract alsocontains rhamnose, mannose, and glucosamine. In inhibi-tion-blocking studies, only glucose significantly inhibitedmonoclonal antibody 8A.The amino acid composition of purified antigen 227 recog-

nized by antibody 8A also differs from that of the methanolextract. The purified antigen contains less glutamic acid andmore serine and glycine. The high content of glutamic acid inthe methanol extract is not surprising. High concentrations

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ethanosarci=a barkeri strain 227

+ 2 x methanol

1500 x g30 min

Supernatant (Ag+)

25,000 x g120 min

Supernatant (Ag+)Antigen 227 methanol

extract

2 x silt-methanolprecipitation

Pellet (Ag-)(discard)

Pellet (Ag-)(discard)

25,000 x g60 min

Supernatant (Ag-)

Step 4

Precipitate (Ag+)+ H20

sephadexG 25

High MW fractions (Ag+)(pool)

Step 5

Low MW fractions(Ag-)

8Asepharose4B

Bound fraction (Ag+) Non-bound fraction (Ag-)Purified antigen 227

FIG. 3. Protocols for the methanol extract and purified antigen 227 preparations. Ag+ or Ag- indicates that antigen 227 was detected or

not detectable, respectively, by slide immunoenzymatic assay with monoclonal antibody 8A.

of glutamate were already reported in M. barkeri MS, whereit plays an important role in the formation of other aminoacids via transamination (14).Some observations suggest that antigen 227 is associated

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FIG. 4. Affinity chromatography of partially purified antigen 227,eluted with distilled water (arrow 1) and 5% NH40H (arrow 2).

with the lipid structure. This antigen can be solubilized very

efficiently from whole cells by methanol, and it appears onlyin the organic phase after chloroform-methanol extraction.However, we were unable to demonstrate lipid in ourpreparations by classical procedures, which may not havebeen sufficiently sensitive or technically appropriate to de-tect the kind of lipid present in methanosarcinae. Alterna-tively, antigen 227 may form an association with a separatelipid structure.

In addition to antigen 227, two fractions containing aminoacids were found in the methanol extract by Sephacryl S300chromatography. The origin and locations of these fractionsare uncertain, since none of the monoclonal antibodiesrecognized them in our assays. This investigation will becontinued with other antibodies in our panel.Our data also suggest the possibility that the M. barkeri

227 cell envelope contains a glucose homopolysaccharide.Analysis of cell envelopes of Methanosarcina mazei dis-closed the presence of a matrix of loose fibrils, whichsurrounds individual cells (21). However, single cells arecapable of growing without this envelope, which seems to berequired for the formation of the sarcinal colonies.

Step 1

Step 2

Step 3

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Galacturonic acid and N-acetyl galactosamine have beenreported as principal components of this matrix; Glucosewas also present, although in much lesser quantity. Glucosehas been found in the cell wall of another archaebacterium,Halobacterium halobium Ml, and an asparagine-glucoselinkage has been indicated (12, 27, 28). Whether this kind oflinkage also occurs in M. barkeri is an interesting questionfor future study.

ACKNOWLEDGMENTSBacteria were provided by T. L. Miller and M. J. Wolin and their

collaborators, whom we thank. We also thank Anthony L. Tarentinofor advise and critical reading of the manuscript.

This work was supported in part by the U.S. Department ofEnergy Grant DE-ACO2-81ER10880 and DE-FG02-84ER13197 andby North Atlantic Treaty Organization (NATO) Research Grant261.80-81. J.C.G. was a fellow of CONICET, Republica Argentina.

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3. Bureau, D., and J. Daussant. 1983. Desorption following im-munoaffinity chromatography: generalization of a gentle proce-dure for desorbing antigen. J. Immunol. Methods 57:205-213.

4. Conway de Macario, E., A. J. L. Macario, and R. J. Jovell. 1983.Quantitative slide micro immunoenzymatic assay (micro-SIA)for antibodies to particulate and non-particulate antigens. J.Immunol. Methods 59:39-47.

5. Conway de Macario, E., A. J. L. Macario, and 0. Kandler. 1982.Monoclonal antibodies for immunochemical analysis of meth-anogenic bacteria. J. Immunol. 129:1670-1674.

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7. Dubois, M., K. A. Gilles, J. K, Hamilton; P. A. Rebers, aid F.

Smith. 1956. Colorimetric method for determination of sugarand related substances. Anal. Chem. 28:350-356.

8. Ey, P. L., S. J. Prowse, and C. R. Jenkin. 1978. Isolation of pureIgGl, IgG2a and IgG2b immunoglobulins from mouse seirumusing protein A-Sepharose. Immunochemiistry 15:429-436.

9. Ferguson, T. J., and R. A. Mah. 1983. Isolation and character-ization of .an H2-oxidiiing thermophilic metharnogen. Appl.Environ. Microbiol. 45:265-274.

10. Fox, G. F., L. W. Magrum, W. E. Balch, R. S. Wolfe, and C. R.Woese. 1977. Classification of methanogenic bacteria by 16Sribosomal RNA characterization. Proc. Natl. Acad. Sci. USA74:4537-4541.

11. Honda, S., K. Kakimoto, K. Kakehi, and K. Takiura. 1974.Fluorometric determination of reducing sugars withethylandiamine sulfate. Anal. Chim. Acta 70:133-139.

12. Kaindler, 0. 1982. Cell wall structure and their phylogeneticimplications. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig.Reihe C 3:149-160.

13. Kandler, O., and H. Kohig. 1985. Cell envelopes of Ar-chaebacteria, p. 413-457. In C. R. Woese and R. S. Wolfe(ed.), The bacteria: a treatise on structure and function. Aca-

demic Press, Inc., Orlando.14. Kenealy, W. R., T. E. Thompson, K. R. Schubert, and J. G.

Zeikus. 1982. Ammonia assimilation and synthesis of alanine,aspartate, and glutamate in Methanosarcina barkeri andMethanobacterium thermoautotrophicum. J. Bacteriol. 150:i357-1365.

15. Keth, A. B., J. M. Kemire, L. E. E. Cannon, and H. 0,Halverson. 1980. In vitro synthesis of repressible yeast acidphosphatase: identification of multiple mRNAs and products.Proc. Natl. Acad. Sci. USA 77:4504-4508.

16. Macario, A. J. L., and E. Con*vay de Macario. 1983. Antigenicfingerprinting of methanogenic bacteria with polyclonal anti-body probes. Syst. Appl. Microbiol. 4:451-458.

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