JOURNAL OF BACFERIOLOGY, Nov. 1974, p. 945-954Copyright i 1974 American Society for Microbiology

Vol. 120, No. 2Printed in U,S.A.

Protein and Carbohydrate Composition of the Cell Envelope ofHalobacterium salinarium'

M. F. MESCHER, J. L. STROMINGER, AND S. W. WATSONThe Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138, and Woods Hole

Oceanographic Institution, Woods Hole, Massachusetts 02543

Received for publication 13 May 1974

The isolated cell envelope of Halobacterium salinarium strain 1 contained 15to 20 proteins that were resolved by polyacrylamide gel electrophoresis in thepresence of sodium dodecyl sulfate. All but one of these proteins had molecularweights of 130,000 or less and together accounted for 50 to 60% of the totalenvelope protein. The remaining 40 to 50% of the envelope protein was accountedfor by a single protein with an apparent molecular weight of approximately194,000 that stained for carbohydrate with periodate-Schiff reagent. Theproteolytic enzymes trypsin and Pronase were used to show that the carbohy-drate is covalently bound to the protein. Separation of amino sugar- andhexose-containing tryptic peptides by gel filtration indicated that all of thenonlipid carbohydrate of the cell envelope is covalently bound to protein. Theresults of partial purification by phenol extraction indicated that both the aminosugar and hexose are bound to the 194,000-molecular-weight protein. Exposure ofisolated cell envelopes to low salt concentration resulted in solubilization of amajority of the envelope proteins. A relatively small number of proteins,including the high-molecular-weight, carbohydrate-containing protein, remainedbound to the sedimentable cell membrane fraction.

The obligately halophilic bacteria of thegenus Halobacterium lack the peptidoglycanlayer characteristic of most bacteria. Althoughthe cell envelopes of the Halobacteria possess anamino sugar-containing component, neithermuramic acid, diaminopimelic acid (8), norD-alanine (unpublished observation) has beenfound. Despite this lack of a rigid peptidoglycanlayer, the organisms are able to maintain arod-shaped morphology when grown under opti-mal conditions. Early studies of the envelopes ofthe Halobacteria failed to demonstrate thepresence of a cell wall external to the plasmamembrane (8). Improved methods of specimenpreparation have shown, however, that theorganisms do possess an external cell wall layer(2, 20, 21), and studies of H. salinarium bySteensland and Larsen (20), H. halobium byStoeckenius and Rowen (21) and Marshall et al.(10), and H. cutirubrum by Kushner et al. (7)have shown that this layer is composed largelyof protein.A number of studies have been done on the

effects of ion concentration on the structuralstability of the cell envelopes of these orga-

IContribution no. 3356 from the Woods Hole Oceano-graphic Institution.

nisms. The morphological changes and ultimatelysis of the cells that occur upon lowering thesalt concentration of their suspending mediumare not simply a result of osmotic imbalance,since similar morphological changes are seen inintact cells and in isolated cell walls. In thethree halophiles that have been most exten-sively studied, H. salinarium (20), H.cutirubrum (7), and H. halobium (2, 21), agradual lowering of the salt concentration re-sults in a conversion from rods to sphericalforms. This morphological change is accompa-nied by a breakdown in the structure of theouter envelope layer, resulting in disappearanceof the patterned surface layer and a frayedappearance of the cell surface when seen in thinsection. Similar structural changes are seen incell envelopes prepared at high salt concentra-tions and resuspended in solutions of lower saltconcentration. These changes in isolated cellenvelopes are accompanied by the release ofprotein into solution. Further lowering of thesalt concentration results in complete dissolu-tion of the envelope, leaving only membranefragments that have lost much of their protein.Although the overall analysis of the envelopes

of these organisms has been determined and the945

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MESCHER, STROMINGER, AND WATSON

effects of salt concentration on their morphol-ogy and composition have been studied, theirprotein composition has not been examined norhas the nature of the amino sugar- and hexose-containing components been determined. Dur-ing studies of cell envelopes of H. salinarium, itwas found that the amino sugar behaved as if itwere protein bound. This finding, together withthe unusual nature of Halobacterium cell enve-lopes, prompted a study of the protein composi-tion of the cell envelopes of H. salinarium andexamination in more detail of the nature of thecarbohydrate-containing component(s).

(This paper was presented in part at the 73rdAnnual Meeting of the American Society forMicrobiology, 6-13 May 1973, Miami Beach,Fla.).

MATERIALS AND METHODSGrowth and harvesting. H. salinarium strain 1

(ATCC 19700) was grown in liquid culture in themedium described by Steensland and Larsen (20)with the exceptions that yeast extract (Difco) wassubstituted for yeast autolysate and the sodiumchloride used was reagent grade (Fisher). The bacte-ria were grown at 37 C in 1-liter shake culturesinoculated with 50 ml of a 2-day shake culture andharvested at the end of log phase (38 to 42 h) in arefrigerated centrifuge. Uniformly labeled cell enve-lope proteins were obtained by growing cells in themedium described above, to which 150 ACi of mixed14C-labeled amino acids (New England NuclearCorp.) per liter had been added.

Cell envelope preparation. After harvesting, cellswere washed twice in basal salts solution having thesame composition as the growth medium: 25% (wt/vol) NaCl, 1.0% (wt/vol) MgSO,-7H20, 0.02% (wt/vol) CaCl2.2H,O, and 0.5% (wt/vol) KCl. Washingand all subsequent operations were done at 0 C. Thewashed wet-cell pellet was suspended in 3 volumes ofbasal salts and 2 volumes of acid-washed, 120-,gmglass beads (3-M Co.) and ground for 5 min in aMini-mill (Gifford-Wood Co.). The broken-cell sus-pension was then centrifuged at 4,500 x g for 10 minto remove glass beads, debris, and whole cells. Thesupematant solution was centrifuged for 40 min at100,000 x g. The resulting envelope pellet was thenwashed twice by resuspending in 50 volumes of basalsalts and again centrifuged at 100,000 x g for 40 min.The washed cell envelope pellet was stored at - 70 C ifnot used immediately.

Quantitative determinations. Salt-free dryweights were determined by drying samples to con-stant weight at 105 C and subtracting the ash remain-ing after heating at 1,800 C for 2 h. Lipids wereextracted by suspending envelopes in 10 ml of 2:1chloroform-methanol per g of wet cell envelopes andmixing with a Vortex mixer. The suspension wasallowed to stand for 15 min at room temperature withoccasional mixing and then centrifuged. The pelletwas reextracted twice in the same manner and then

dried by lyophilizing. The supernatant solutions werecombined and evaporated to dryness for determina-tion of cell envelope lipid content.

Protein was determined by the method of Lowry etal. (9), with bovine serum albumin as standard.Neutral hexoses were determined by the anthroneassay, using the procedure described by Spiro (19)with glucose as the standard. Hexosamine was deter-mined by a modification of the Morgan-Elson assay(4), with glucosamine-hydrochloride as the standard.

Two-dimensional thin-layer chromatography ofamino acids and amino sugars was done on Silica GelG plates by using chloroform-methanol-17% ammo-nium hydroxide (2:2:1, vol/vol) in the first directionand phenol-water (75:25, wt/wt) in the second direc-tion (14). Amino acids were detected by spraying withninhydrin and amino sugars by the method of Par-tridge (13), using p-dimethylaminobenzaldehyde.

Protease treatment. Proteolytic digestion of enve-lope protein was done by using trypsin (Calbiochem)in 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 8.2, at an enzyme to enve-lope protein ratio of 1:10 and Pronase (Calbiochem) in0.01 M Tris-hydrochloride buffer, pH 7.8, at anenzyme to envelope protein ratio of 1:10. Mixtureswere incubated at 37 C for 24 to 48 h with 0.02%sodium azide added to prevent bacterial growth.

Gel filtration. Gel filtration of trypsin-digested cellenvelope protein was done on a column (2.5 by 70 cm)of Sephadex G-100 (Pharmacia), using 10 mM ammo-nium bicarbonate, pH 7.0 (with 0.02% sodium azideadded), for elution. Fractions of 10 ml were collectedand assayed for absorbance at 280 nm, amino sugar,and neutral hexose.

Polyacrylamide gel electrophoresis in sodiumdodecyl sulfate. Sodium dodecyl sulfate (SDS)-gelelectrophoresis was done by the procedure of Weberand Osborn (22). Protein was visualized with Coomas-sie brilliant blue and carbohydrate by the periodate-arsenite-Schiff method (PAS stain) of Fairbanks et al.(3). Samples were dissolved in 0.01 M sodium phos-phate buffer, pH 7.2, containing 1% SDS, 0.14 M2-mercaptoethanol, 10% glycerol, and 0.002% bromo-phenol blue, placed in a boiling water bath for 10 min,cooled to room temperature, and applied to the gel.For determination of the radioactivity profile oflabeled gels, they were cut into 1-mm slices and theslices were shaken overnight at 30 C in 10 ml oftoluene scintillation fluid containing 3% Protosol(New England Nuclear Corp.) before counting.

RESULTS

Preparation of H. salinarium cell envelopesby mechanical disruption in a salt solutionhaving the same ionic composition as thegrowth medium results in the isolation of enve-lopes in the form of closed vesicles. Cell enve-lope fragments prepared in this manner retaintheir outer wall layer as evidenced by thepresence of the hexagonal surface patterns inthese preparations (20). The cell envelope ac-

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counts for approximately 20% of the salt-freedry weight of the whole cell. Analysis of theenvelopes (Table 1) gave values for protein,lipid, and carbohydrate similar to those foundby Steensland and Larsen (20). As in theirpreparations, spectrophotometric data indi-cated that the material unaccounted for isnucleic acid. Lipid extraction was found toremove approximately 25% of the neutral hex-ose present in the envelopes. Glycolipid hasbeen reported in H. cutirubrum (6), and itappears that approximately one-quarter of thehexose in H. salinarium envelopes is in the formof glycolipid. All of the amino sugar remained inthe lipid-insoluble fraction.

Lipid-extracted cell envelopes were solubi-lized by boiling in 1.0% SDS and 0.14 M2-mercaptoethanol. The resulting material was

electrophoresed on SDS gels and stained withCoomassie blue. Approximately 15 to 20 proteinbands were observed (Fig. 1A). The majority ofthese proteins had molecular weights of 130,000or less, similar to the findings of Schnaitman(16) for Escherichia coli envelopes, in which allof the proteins were found to have molecularweights of less than 120,000. Two of the bands,however, had lower mobilities than f,-galactosi-dase (molecular weight, 130,000), the major one

being a broad band having the same mobility as

myosin (molecular weight, 194,000). When an

identical gel was stained for carbohydrate withthe PAS reagent, two bands were seen (Fig. 1B),a broad band with the same mobility as myosinand a more faintly staining, lower-molecular-weight band. The mobilities of these bandscorresponded to those of the two high-molecu-lar-weight, Coomassie blue-staining bands. Ifthe envelopes were solubilized directly in SDS,without lipid extraction, a third PAS-positiveband appeared having approximately the same

mobility as the bromophenol blue dye marker.This material was apparently the glycolipidpresent in the envelope.

It was observed that the age and prior treat-ment of the envelope preparation caused a

change in the relative intensity of the twohigh-molecular-weight bands as well as theoccasional appearance of lower-molecular-weight, PAS-positive bands, suggesting that theenvelope preparations contain a contaminatingprotease activity. To examine this possibility,envelopes were prepared in the usual manner

and then suspended in either distilled water or

basal salts and incubated for varying periods oftime at 37 C. After the incubation period, SDSwas added to a final concentration of 1% and2-mercaptoethanol was added to 0.14 M. The

sample was placed in a boiling water bath for 10min before being frozen and stored. When all ofthe samples had been prepared, portions con-taining equal amounts of protein were run inparallel on SDS gels and stained with the PASreagent (Fig. 2). It is readily apparent that thehigh-molecular-weight component was beingdegraded with a resulting increase in all of thelower-molecular-weight PAS bands with in-creasing incubation time, including an increasein the amount of the minor PAS-positive bandseen in Fig. 1B. Furthermore, the pattern ofdegradation products observed depended uponthe salt composition of the incubation medium.At low salt concentration there was a moreheterogeneous distribution of products, suggest-ing that more sites for proteolysis had beenexposed either due to an unfolding of theprotein or to the loss of the structural integrityof the envelope, or both. Coomassie blue-stained gels of identical samples showed nonoticeable alteration from the initial patternobserved, except for a decrease in intensity ofthe highest-molecular-weight protein and anincrease in the intensity of the next-highest-molecular-weight component (corresponding tothe minor PAS-positive band). This result maybe due to the greater probability of the high-molecular-weight protein being degraded by aprotease because of its large size and highconcentration relative to the other envelopeproteins (see below). Halobacteria are known toproduce extracellular proteases (11), and itappears likely that isolation of the envelopesresults in sufficient disruption of their struc-tural integrity to make them susceptible tothese proteases. However, the possibility thatthe observed degradation may be due to glycosi-dase activity is not ruled out. In either case, theresults suggest that the cell envelopes of intactH. salinarium cells possess a single high-molecular-weight, carbohydrate-containingprotein; the minor PAS-positive protein is prob-ably a degradation product.

TABLE 1. Cell envelope analysisa

Amt in cell envelope

Substance mgpe 100 mg% remainingmgrperi100tm after lipid

(drywight) extraction

Protein ............. 63 98Hexose ............. 3.6 74Amino sugar ........ 0.6 95Lipid .............. 21 0

aValues are averages obtained from two separatecell envelope preparations.

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A BFIG. 1. SDS gels (5% acrylamide) of envelope proteins (600 Ag per gel). (A) Coomassie blue stained-gel; (B)

PAS-stained gel (arrow on right indicates position of minor PAS-staining band). The positions of standardproteins run on parallel gels are indicated: (1) myosin (194,000); (2) j6-galactosidase (130,000); (3) bovine serumalbumin (69,000); and (4) aldolase (40,000).

To obtain an estimate of the contribution ofthis high-molecular-weight component to thetotal envelope protein, cells were grown in 1 literof medium containing 150 gCi of "4C-labeledamino acids. The "IC-labeled amino acid mix-

ture was added to the normal growth mediumbefore inoculation. Inoculation, growth, andharvesting were carried out as described inMaterials and Methods. Approximately 30% ofthe added radioactivity was incorporated, and

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CELL ENVELOPE COMPOSITION OF H. SALINARIUM

.t

.X

A0 hr.

B1.5 hr.

loxI,

Di'ox

16 hrs.No Salt

Icm cm

FIG. 2. Effect of incubation at 37 C on carbohydrate-containing cell envelope proteins. Scans at 560 nm ofPAS-stained SDS gels (7.5% acrylamide) a&e shown. The regions of the gels to the right of the dotted line werescanned at 10 times greater sensitivity then the high-molecular-weight band. Envelopes (A) immediately afterpreparation; (B) after incubation for 1.5 h in basal salts; (C) after incubation for 16 h in basal salts; (D) afterincubation for 16 h in distilled water.

26% of the incorporated activity was present inthe isolated cell envelopes. After lipid extrac-tion, the envelopes were run on SDS gels, sliced,and counted (Fig. 3A). Parallel gels containingan equal amount of protein were stained withCoomassie blue (Fig. 3B) and PAS reagent (Fig.3C). All of the radioactivity was recovered fromthe gel except for approximately 5% remainingat the top of the gel. Because of the relativelylow specific activity (3.6 x 104 counts/min permg) of proteins labeled in this manner, theresolution of the lower-molecular-weight com-ponents was poor. The high-molecular-weightPAS-positive band was well resolved, however,and accounted for 40 to 50% of the radioactivityon the gel. (The "4C-labeled, carbohydrate-con-taining protein has subsequently been purifiedand found to have the same specific activity asthe whole envelope protein; therefore, it is notpreferentially labeled.) Comparison of thesliced gel and the Coomassie blue-stained gelindicates that the high-molecular-weight bandstained very poorly with Coomassie blue incomparison with the lower-molecular-weight

proteins. A similar phenomenon is observedwith the acidic glycoprotein of erythrocytemembranes (3).To determine whether the carbohydrate por-

tion of the high-molecular-weight component is,in fact, covalently bound to protein, lipid-extracted envelopes were treated with the pro-teases, trypsin, and Pronase. After extensivedigestion with trypsin, a single, low-molecular-weight, PAS-positive band was seen on gels(Fig. 4). It also stained lightly with Coomassieblue and was the only Coomassie blue-positiveband present. Treatment of the trypsin-digested material (or of lipid-extracted enve-lopes) with Pronase further degraded it so thatno PAS-positive band was found on the gel (Fig.4).The trypsin degradation products were fur-

ther examined by gel filtration. Before trypsindigestion, all of the nonlipid carbohydrate (bothhexose and amino sugar) of cell envelopessolubilized in 10 mM ammonium bicarbonateran as a single peak in the excluded volume on aSephadex G-100 column. After trypsin degrada-

C 16 hrs.loxI1

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MESCHER, STROMINGER, AND WATSON

A Phosphorylose A/3- golactosidose

_ Myosin BSA Ajdolose

400-

300-

20-1

d.) 200-41 Bram-Phenol Blue

100-'

0 2 4 6

cm

BI_ l. it .A

_ _ _

*

C

FIG. 3. SDS gels (5% acrylamide) of "IC-labeled cell envelopes (lipid free). Gels were run in parallel withequal amounts of protein (120 pg) applied to each. Positions of standard proteins run in parallel are shown byarrows. (A) Radioactivity profile of sliced gel; (B) Coomassie blue-stained gel; (C) PAS-stained gel.

tion, the profile shown in Fig. 5 was obtained.The hexose remained in the excluded peak andthe amino sugar was found in a single, includedpeak. SDS-gel electrophoresis of the material inthe hexose peak resulted in a single PAS-posi-tive band having the same mobility as thePAS-positive band seen in the tryptic digest ofwhole-cell envelope protein (Fig. 4A). No bandswere seen on SDS gels of the material in theamino sugar peak, probably because of the lowmolecular weight of the peptide. Two-dimen-sional thin-layer chromatography (see Mate-rials and Methods) of acid hydrolysates (6 NHCl for 16 h at 105 C, in vacuo) of pooled aminosugar- and pooled hexose-containing fractionsindicated that most of the common amino acidswere present in both. Two amino sugars were

also detected in the amino sugar peak by boththe ninhydrin and p-dimethylaminobenzalde-

hyde sprays, one having mobilities identical tothose of glucosamine. The other ran in theregion of galactosamine and mannosamine,which were not resolved in the system used. Thesusceptibility of the carbohydrate-containingmaterial to trypsin suggests that all of thenonlipid carbohydrate of the cell envelope of H.salinarium was covalently linked to protein.The phenol extraction procedure originally

used by Westphal et al. (23) for extraction oflipopolysaccharide from envelopes of gram-neg-ative bacteria has been used by a number ofinvestigators for isolation of cell surface glyco-proteins from erythrocyte membranes (5). Itwas found to be effective for obtaining partialpurification of the carbohydrate-protein com-plex of H. salinarium cell envelopes. The ex-traction was carried out by using an equalvolume of 0.05 M Tris-hydrochloride buffer, pH

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aqueous phase along with approximately 75% ofthe neutral hexose. SDS gels of the two phases(after dialysis to remove the phenol) show thatthe only major protein present in the aqueousphase was the high-molecular-weight, carbohy-drate-containing protein (and its proteolyticdegradation products) (Fig. 6C and C'). All ofthe remaining envelope proteins were recoveredin the phenol phase (Fig. 6B). Trypsin digestionof the material recovered in the aqueous phaseresulted in amino sugar- and hexose-containingpeptides eluting at the same positions on aSephadex G-100 column and having identicalmobilities and staining properties on SDS gelsas those observed for whole-envelope digests.Co-purification of the amino sugar and hexosemoieties suggested that both are bound to thehigh-molecular-weight protein.Lowering of the salt concentration of the

suspending medium results in release of proteinfrom isolated cell envelopes of H. salinarium(20). Steensland and Larsen (20) have shownthat exposure of cell envelopes to low saltconcentration followed by centrifugation resultsin the isolation of an envelope fraction that ismissing the outer layer, and the only organizedstructure that appears to remain is the cellmembrane. This cell membrane fraction isconsiderably enriched in lipid by comparisonwith the intact cell envelope. To determinewhether all of the envelope proteins are solubi-lized to an equal extent under these conditionsor whether a specific group of proteins remainbound to the sedimentable membrane fraction,cell envelopes were prepared in the usual man-ner and the wet pellet was suspended in dilutesalt solution to give a final concentrationof approximately 0.14 M NaCl, 5 mMMgSO,.7H2O, and 10 mM KCl. The resus-

A BFIG. 4. SDS gels (5% acrylamide) of cell envelope

protein after incubation with added proteases. Stain-ing was done with the PAS reagent. (A) Trypsin-digested whole-cell envelopes; (B) Pronase-digestedwhole-cell envelopes.

7.2, containing 0.5% NaCl and buffer-saturatedphenol and was equally effective when done for30 min at either room temperature or at 85 C.All of the amino sugar was recovered in the

Fraction No.

FIG. 5. Elution profile on Sephadex G-100 (2.5 by70 cm) of whole-envelope protein (600 mg) aftertrypsin digestion. Samples of fractions were assayedfor hexose (absorbancy at 620 nm), amino sugar(absorbancy at 585 nm), and absorbancy 280 nm.

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A w

A

U_

B C C'IFIG. 6. Phenol extraction of cell envelopes. SDS gels (5% acrylamide) of: (A) whole envelopes (800 Mg of

protein) (Coomassie blue stained); (B) phenol phase after phenol extraction (400 ,g of protein) (Coomassie bluestained); (C) aqueous phase after phenol extraction (400 Mg of protein) (Coomassie blue stained); (C') aqueousphase after phenol extraction (400 usg of protein) (PAS stained; arrow on right indicates position of minorPAS-staining band).

pended cell envelopes were stirred for 5 min andthen centrifuged at 100,000 x g for 1 h. Spectro-photometric scans of Coomassie blue-stainedgels of whole-cell envelopes (Fig. 7A) and of thepellet obtained after exposure to low salt con-centration (Fig. 7B) showed that the materialremaining sedimentable under the above condi-tions was greatly enriched in several of theenvelope proteins, among them the high-molecular-weight, carbohydrate-containingprotein. The majority of the envelope proteinsremained in very low amounts, if at all, in thesedimentable cell membrane fraction relative totheir concentration in whole cell envelopes.

DISCUSSIONCell envelopes of Halobacterium salinarium

lack the peptidoglycan layer and lipopolysac-

charide-containing outer membrane componentnormally found in cell envelopes of gram-nega-tive bacteria but have been shown to possess anexternal cell wall layer in addition to a plasmamembrane with a typical bilayer appearance(20). When the proteins of the cell envelope areexamined by SDS-gel electrophoresis, the pat-tern obtained resembles in some respects thatseen for E. coli (16, 17) and other gram-negativebacteria (15). Approximately 50% of the totalenvelope protein is present in 15 to 20 bandshaving molecular weights of 130,000 or less. Thestrikingly different feature of the protein com-position of these envelopes is the absence of amajor band at 42,000 daltons, which in E. coliaccounts for approximately 40% of the envelopeprotein and which Schnaitman (17) has shownto be a group of at least three polypeptidesassociated with the outer membrane. Instead,

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the remaining 40 to 50% of the protein in H.salinarium envelopes is accounted for by a

protein with a mobility on SDS gels correspond-ing to a molecular weight of approximately194,000. This protein has both all of the aminosugar and all of the nonlipid hexose covalentlybound to it as indicated by PAS staining, itssusceptibility to proteases, and its behavior on

phenol extraction. SDS gels are known to giveanomalous molecular weight values for proteinscontaining substantial amounts of carbohydrate(18), and the value of 194,000 may therefore be

Myosin S-got BSA 0-3-P DH4m(194,000) (13 ( (36000)

I

High SaltEnvelope"A

Low Salt"Membrane"

B

cmFIG. 7. Effect of low salt concentration on envelope

protein content. Scans at 620 nm of Coomassieblue-stained SDS gels are shown. (A) Whole-cellenvelopes; (B) sedimentable envelope fraction aftersuspension in medium of low salt concentration.

in error. However, this protein would containonly approximately 12% carbohydrate (calcu-lated from the data of Table 1 and Fig. 3). Thisrelatively low carbohydrate content would notbe expected to result in a large error in themolecular weight as determined by SDS gels.The results obtained by SDS-gel electrophoresisdo not rule out the possibility that the high-molecular-weight band is actually two or moreproteins with similar molecular weights. Thus,although both the amino sugar and hexosemoieties are covalently linked to protein, it isnot clear whether they are bound to the sameprotein. The protein runs as a broad band ongels loaded with sufficient material to allowadequate visualization of it and of the lower-molecular-weight envelope proteins by Coomas-sie blue (Fig. 1A and 6) but is sharpenedconsiderably when lesser amounts are electro-phoresed (Fig. 3A). Furthermore, if there ismore than one PAS-staining protein, they copu-rify upon phenol extraction. Further purifica-tion and study of the high-molecular-weightprotein will be necessary to resolve this ques-tion.

Several bacterial envelope proteins havingcovalently bound carbohydrate have been re-ported, and two types of linkage between theprotein and the carbohydrate-containingmoiety have been found. Braun and Bosch (1)have demonstrated the occurrence of a lipo-protein bound through the 6-amino group of itsC-terminal lysine to the diaminopimelic acid ofthe peptidoglycan in E. coli. Wober and Alau-povic (24), working with Serratia marcescens,and Wu and Heath (25), working with E. coli,have shown that most of the lipopolysaccharideof these organisms is present as a protein-lipopolysaccharide complex covalently linkedby a phenol-sensitive bond. In light of theabsence of peptidoglycan and lipopolysaccha-ride and its stability in hot phenol, the carbohy-drate-containing protein of H. salinarium ap-pears to resemble neither of these molecules.Okuda and Weinbaum (12) have reported thepresence of phenol-extractable "glycoproteins"in the cell envelope of E. coli. They have shownthat the carbohydrate-containing moiety is co-valently bound to protein but have not demon-strated a direct linkage between a carbohydratemolecule and an amino acid as occurs in plantand animal glycoproteins; the question ofwhether E. coli or other bacteria possess glyco-proteins of the type found in higher organismsthus remains open.The high-molecular-weight, carbohydrate-

containing protein of H. salinarium is beingstudied further in order to determine the nature

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of the linkage between the carbohydrate moietyand the protein and to attempt to determine itsfunction. The large proportion of the high-molecular-weight protein in the cell envelope ofH. salinarium suggests that it plays an impor-tant role in the structure of the envelope, e.g., inits shape. Unlike the majority of envelopeproteins, it remains bound to the lipid-enrichedcell membrane at low salt concentration andmay serve as a bridge between the membraneand the less firmly bound envelope proteins. Inaddition to further study of the H. salinariumcarbohydrate-protein complex, H. cutirubrumand H. halobium cell envelopes are being exam-ined to determine whether they have similarcarbohydrate-containing proteins. The cell en-velopes of these bacteria have amino sugar andhexose compositions similar to those of H.salinarium, and a preliminary report on theoccurrence of protein-bound hexose in the cellenvelope of H. halobium has been presented(M. A. Koncewicz, Biochem. J. 128:124, 1972).

ACKNOWLEDGMENTSThis investigation was supported by Public Health Service

research grants AM-13230, from the National Institute ofArthritis, Metabolism, and Digestive Diseases, andGM-11214, from the National Institute of General MedicalSciences, and by grant GB-29747 from the National ScienceFoundation.

LITERATURE CITED

1. Braun, V., and V. Bosch. 1972. Sequence of the murein-lipoprotein and the attachment site of the lipid. Eur. J.Biochem. 28:51-69.

2. Cho, K. Y., C. H. Doy, and E. H. Mercer. 1967.Ultrastructure of the obligate halophilic bacteriumHalobacterium halobium. J. Bacteriol. 94:196-201.

3. Fairbanks, G., T. L. Steck, and D. F. H. Wallach. 1971.Electrophoretic analysis of the major polypeptides ofthe human erythrocyte membrane. Biochemistry10:2606-2617.

4. Ghuysen, J.-M., D. J. Tipper, and J. L. Strominger. 1966.Enzymes that degrade bacterial cell walls, p. 685-699.In E. F. Neufeld and V. Ginsberg (ed.), Methods inenzymology, vol. 8. Academic Press Inc., New York.

5. Howe, C., K. 0. Lloyd, and L. T. Lee. 1972. Isolation ofglycoprotein from red cell membranes using phenol, p.236-245. In V. Ginsburg (ed.), Methods in enzymology,vol. 28. Academic Press Inc., New York.

6. Kates, M., B. Palameta, M. P. Perry, and G. A. Adams.1967. A new glycolipid sulfate ester in Halobacteriumcutirubrum. Biochim. Biophys. Acta 137:213-216.

7. Kushner, D. J., S. T. Bayley, J. Boring, M. Kates, and N.E. Gibbons. 1964. Morphological and chemical proper-ties of cell envelopes of the extreme halophile, Halobac-terium cutirubrum. Can. J. Microbiol. 10:483-497.

8. Larsen, H. 1967. Biochemical aspects of extreme halo-philism. Advan. Microb. Physiol. 1:97-132.

9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

10. Marshall, C. L., A. J. Wicken, and A. D. Brown. 1969.The outer layer of the cell envelope of Halobacteriumhalobium. Can. J. Biochem. 47:71-74.

11. Norberg, P., and B. V. Hofsten. 1969. Proteolytic en-zymes from extremely halophilic bacteria. J. Gen.Microbiol. 55:251-256.

12. Okuda, S., and G. Weinbaum. 1968. A membrane-specific glycoprotein from Escherichia coli B. Biochem-istry 7:2819-2825.

13. Partridge, S. M. 1948. Filter-paper partition chromatog-raphy of sugars. I. General description and applicationto the qualitative analysis of sugars in apple juice, eggwhite and foetal blood of sheep. Biochem. J.42:238-248.

14. Randerath, K. 1968. Thin layer chromatography, p.110-115. Academic Press Inc., New York.

15. Schnaitman, C. A. 1970. Comparison of the envelopeprotein compositions of several gram-negative bacteria.J. Bacteriol. 104:1404-1405.

16. Schnaitman, C. A. 1970. Examination of the proteincomposition of the cell envelope of Escherichia coli bypolyacrylamide gel electrophoresis. J. Bacteriol.104:882-889.

17. Schnaitman, C. A. 1973. Outer membrane proteins of E.coli. H. Heterogeneity of major outer membrane poly-peptides. Arch. Biochem. Biophys. 157:553-560.

18. Segrest, J. P., and R. L. Jackson. 1972. Molecular weightdetermination of glycoproteins by polyacrylamide gelelectrophoresis in sodium dodecyl sulfate, p. 54-63. InV. Ginsburg (ed.), Methods in enzymology, vol. 28.Academic Press Inc., New York.

19. Spiro, R. G. 1966. Analysis of sugars found in glyco-proteins, p. 3-26. In E. F. Neufeld and V. Ginsburg(ed.), Methods in enzymology, vol. 8. Academic PressInc., New York.

20. Steensland, H., and H. Larsen. 1969. A study of the cellenvelope of the Halobacteria. J. Gen. Microbiol.55:325-336.

21. Stoeckenius, W., and R. Rowen. 1967. A morphologicalstudy of Halobacterium halobium and its lysis inmedia of low salt concentration. J. Cell. Biol.34:365-393.

22. Weber, K., and M. Osborn. 1969. The reliability ofmolecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem.244:4406-4412.

23. Westphal, O., 0. Luderitz, and F. Z. Bister. 1952. Uberdie Extraktion von Badterien mit Phenol/Wasser. Z.Naturforsch. 7b:148-155.

24. Wober, W., and P. Alaupovic. 1971. Studies on theprotein moiety of endotoxin from gram-negative bacte-ria. Characterization of the protein moiety isolated byphenol treatment of endotoxin from Serratiamarcescens 08 and Escherichia coli 014 K85. Eur. J.Biochem. 19:340-356.

25. Wu, M.-C., and E. C. Heath. 1973. Isolation and charac-terization of lipopolysaccharide protein from Esche-richia coli. Proc. Nat. Acad. Sci. U.S.A. 70:2572-2576.

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