Zbl. Bakt. Hyg., I. Abt, Orig. C 3, 171-178 (1982)

1 Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen,2 Lehrstuhl fiir Mikrobiologie, Universitat Miinchen and3 Max-Planck-Institut fiir Immunbiologie, Freiburg, Federal Republic of Germany

Chemical Composition and Structure of the Cell Wall of

Halococcus morrhuae*

K. H. SCHLEIFER 1, J. STEBER 2, and H. MAYER 3

Received July 3, 1981

Summary

The chemical composition of trypsin-treated cell walls of three strains of Halococcusmorrhuae was determined. Neutral sugars (glucose, mannose, galactose), uronic acids(glucuronic and galacturonic acids), amino sugars (glucosamine, galactosamine), gulo­saminuronic acid, acetate, glycine and sulfate were found as major constituents. The cellwall of H. morrhuae CCM 859 was studied in more detail. The major cell wall polymerof this strain is a complex heteroglycan which seems to be responsible for the rigidityand stability of the cell wall. The amino groups of the amino sugars are predominantlyNvacerylatcd. A substitution of the amino groups with glycine instead of acetate couldbe found for part of the glucosamine residues. Sulfate groups are covalently bound asesters to secondary hydroxyl groups in equatorial conformation. Based on periodatecleavage and permethylation studies of the cell wall and analyses of isolated oligo­saccharides, the chemical structure of the cell wall polymer can be proposed as follows:

Sulfate groups are linked to hydroxyl groups in positions 2 and/or 3 of uronic acids,galactose and galactosamine residues. Glucose, galactose, galacturonic acid and all aminosugars are 1 ->- 4 glycosidically linked in the cell wall polymer. A part of the glucose, galac­tose and to a lesser extent mannose residues possess more than two glycosidic linkagesand represent possible branching points. Glycine residues may playa role in connectingglycan strands through peptidic linkages between the amino group of glucosamine andthe carboxyl group of an uronic acid or gulosaminuronic acid.

Key words: Halococcus morrhuae - Cell wall - Heteropolysaccharide - Gules­aminuronic acid

Introduction

The extremely halophilic cocci, Halococcus morrhuae, grow only in the presenceof 2.5 M or higher concentrations of sodium chloride. These red pigmented

':. Paper given at the First International Workshop on Archaebacteria, Miinchen, June 27to July 1, 1981.

12 Zbl. Bakt. Hyg., I. Abt, Orig. C 3

172 K.H.Schleifer, j.Steber, and H.Mayer

cocci have not been studied as extensively as their rod-shaped counterparts, thehalobacteria. In contrast to the latter they are more resistent to osmotic damageand do not lyse in dilute aqueous solutions. This may be due to the differencesin their cell wall structure. Electron microscopic studies of thin sections of H.morrhuae have shown that the cell wall consists of a single homogeneous layerwith a thickness of 50-60 nm (Kocuret aI., 1972). Moreover, studies by Brownand Cho (1970) and Reistad (1972) revealed that the cell walls of these halophiliccocci differ from walls of most other bacteria in lacking muramic acid, the typicalconstituent of peptidoglycan.

Results and Discussion

Chemical analyses of cell walls of three strains of H. morrhuae have shown thatthe major components are carbohydrates (Steber and Schleifer, 1975). Cell wallswere prepared in the same way as those of Gram-positive bacteria by rupturingthe cells with glass beads in a cell mill (Vibrogen Zellmiihle, Biihler, Tiibingen).Crude cell wall preparations were treated with trypsin to remove cell wall proteinsand/or residual membrane proteins which account for 2 Ofo of the dried cell wallmaterial. The removal of the proteins did not diminish the rigidity of the wallsindicating that they are not essential for the structural integrity of the walls. Thequantitative composition of the constituents of purified cell walls is listed inTable 1. The main components are carbohydrates which represent about 60 0 / 0

of the dried cell wall material. Neutral sugars were determined enzymaticallyand by gas chromatography. Glucose, mannose and galactose were found in thecell walls of all threee strains. Glucuronic and galacturonic acids were idendifiedby paper chromatography, paper electrophoresis and gas chromatography. Quan­titative analyses of amino sugars and amino acids were carried out with an aminoacid analyzer. Glucosamine, galactosamine, gulosaminuronic acid and glycine

Table 1. Chemical composition of purified (trypsin-treated) cell walls of three strains ofHalococcus morrhuae

Components pmol/mg cell wallCCM 859 CCM 537 CCM 889

Glucose 0.44 0.39 0.30Mannose 0.35 0.21 0.30Galactose 0.27 0.18 0.17

Total neutral sugars 1.06 0.78 0.77Glucosamine 0.38 0.21 0.35Galactosamine 0.20 0.08 0.24Gulosaminuronic acid 0.11 0.09 0.10

Total amino sugars 0.69 0.38 0.69Uronic acids" 0.67 0.24 0.63Acetate 0.62 n.d, n.d.Glycine 0.1 0.13 0.1Sulfate 1.47 0.8 1.18

" Total uronic acids determined as glucuronic acid. Glucuronic and galacturonic acidswere present in cell walls of CCM 859 in a molar ratio of about 2.3 :1.

Chemical Composition and Structure of the Cell Wall of H. morrhuae 173

were the only ninhydrin-positive components present in acid hydrolysates ofpurified cell walls (Steber and Schleifer, 1975). The occurrence of gulosaminuronicacid in the cell wall of a halococcus was first described by Reistad (1974). Theash content of the cell walls is unusually high and amounts to 23 % of the dryweight. 57 % of the ash consists of sulfate. The main cations are Na", K+, Ca2+ andM g2+.

Cell walls of H. morrhuae CCM 859 were studied in more detail. All effortsto extract single polymers failed (Steber and Schleifer, 1975). The cell wall wasnot degraded by applying 13 different exo- and endoglycosidases. However, cellwall material could be solubilized under milk alkaline conditions (0.5n NaOH,60 DC, 12 h). The solubilized and insoluble material contained the same consti­tuents. It was possible to precipitate solubilized cell wall polymer with a quater­nary ammonium salt such as cetyltrimethylammonium bromide (CTAB) and toredissolve it with KCl-solutions of increasing molarity as a major single fraction(Steber and Schleifer, 1975). The inability to extract single polymers and theprecipitation with CTAB indicate that the cell wall is mainly composed of a singleanionic heteroglycan.

The carboxyl groups of the uronic acids are at least partly unsubstituted. Thiscould be shown from the infrared (IR) spectrum of native cell wall. There wasa strong absorption band at 1610 cm" which is characteristic of carboxylate ions.

The amino groups of the amino sugars are substituted predominantly by acetylgroups and to a lesser extent by glycine residues. This could be demonstratedby negative dinitrophenylation results and by the failure to form anhydrohexosesafter treatment with HN02 (Lagunoff and Warren, 1962). Moreover, cell wallsexamined under mildly acidic conditions to quench the strong signal of thecarboxylate ions show the characteristic amide bands at 1640 and 1550 cm"(Fig. 1 B; Mirelman et aI., 1973). In addition, N-acetylglucosamine and the dini­trophenyl-derivative of N-glycylglucosamine could be isolated from partiallyhydrolyzed cell walls and dinitrophenylated cell walls, respectively (Steber andSchleifer, 1979).

The critical salt concentration for redissolving the cell wall-CTAB complex issimilar to that for mucopolysaccharides such as chondroitin sulfates, namely 0.8molll KCI. Sulfate is the only strongly acidic constituent of the cell wall present

B

A

aoo4000 3000 2000 1600 1200-1wa ve number (e m )

Fig. 1. Infrared spectra of cell walls of H. morrhuae CCM 859. A, untreated cell wall;B, cell wall treated with 0.01 n HCI at room temperature for 1 h.

174 K.H.Schleifer, j.Steber, and H.Mayer

in considerable amounts. This indicates that the cell wall is made up of a sulfatedheteroglycan. The presence of ester bound sulfates was confirmed by IR-spectra.Spectra of cell wall and chondroitin sulfate are quite similar (Fig. 2). In bothpolysaccharides a strong absorption band at 1240 cm! attributable to S = 0 insulfate esters is found. The absorption of the cell wall polymer at 830 cm!indicates the presence of ester sulfate in a secondary equatorial conformation (Royand Trudinger, 1970). The half life of the ester sulfate as estimated from the

A

B

1800 1600 1400 1200

wave number

1000 800

- 1(e m )

600

Fig. 2. Infrared spectra of cell walls of H. morrhuae CCM 859 (A) and chondroitinsulfate (B).

@

GIcos.SOt. 1 At

@l ~Fru-S-P0t. «L

I A 3 I

GalUA

GleN~

At.

eFig. 3. Scheme of a pherogram (paper; pH 1.8) of a partial acid hydrolysate (0.25 moxalic acid, 100°C, 4 h) of cell walls of H. morrhuae CCM 859.

Chemical Composition and Structure of the Cell Wall of H. morrhuae 175

rate of acid hydrolysis also indicates that sulfate is bound to secondary hydroxylgroups in equatorial conformation (Steber and Schleifer, 1975). Because of theacid lability of the sulfate esters rather mildly acidic conditions (0.25 molll oxalicacid, 100 °C, 4 h) had to be applied to isolate sulfated oligosaccharides frompartial hydrolysates of cell walls. The partial hydrolysates were separated bypaper electrophoresis at pH 1.8 (acetic acid/formic acid/H20 = 75: 20.5 : 90.5,X 1) and the acidic fractions were further purified by paper chromatography (Fi­scher and Nebel, 1955). A scheme of a pherogram of a partial acid hydrolysate ofthe cell walls of H. morrhuae CCM 859 is shown in Fig. 3. The main fractionsare A2 and A4. Oligosaccharides containing sulfated galactose and/or uronicacids could be isolated from fractions Al and A2.

Analyses of isolated oligosaccharides obtained after deaminative cleavage ofpartially de-N-acetylated cell walls (20 ml dist. water + 20 ml 33 Ofo acetic acid+ 20 ml 5 Ofo NaN02 + 100 mg de-Nvacerylated cell walls were stirred at roomtemperature for three h) provided more data about the sulfate substitution ofsugars. The sulfate substitution of galactose and uronic acids could be confirmed,whereas that of glucose and glucosamine could be excluded.

Final information about the occurrence of sulfate substituents and the glycosidiclinkages of monomers was obtained by comparative perrnethylarion studies ofintact and completely desulfated cell walls. In addition, carboxyl-reduced cellwall preparations were studied, both before and after desulfation to learn moreabout the glycosidic linkage and sulfate substitution of uronic acids. The resultsobtained from gas liquid chromatography and mass spectrometry of their alditolacetates tHakomori, 1964; Biiimdabl et aI., 1970) are summarized in Table 2.

Table 2. Substitution with sulfate and glycosidic linkage of the different sugars , uronicacids and amino sugars as determined by periodate oxidation, Smith degradation andmethylation studies of intact and desulfated cell wall preparations

Constituent Hydroxyl groupssubstituted byester-bound sulfate

Glycosidic linkage

Glucose

Mannose

Galactose

GlucosamineGalactosamineGulosaminuronic acidGalacturonic acidGlucuronic acid

major part: C-2minor part: C-3

C-3

about 20%: C-3C3(C-4)

major part:minor part : i)

ii)

major part:minor part :about 70% :about 30% :

1~4

1~3+1~2

nonreducingend of thepolysaccharide1 ~2

1 ~3+1~6

1 ~4

1 ~4+1~3

176 K.H.Schleifer, j.Steber, and H.Mayer

Glucose and mannose residues are not sulfated, whereas most of the galactoseresidues are substituted by sulfate esters at the hydroxyl groups of position C-2and/or to a lesser extent at those of C-3. Sulfate esters could also be found inposition C-3 of about 30 Ofo of the galactosamine and 20 'lJ/o of the galacturonicacid residues. All glucuronic acid residues are sulfated in position C-3 and a partof them may even possess a second sulfate substituent in position C-4. All ofthe sulfated substituents are linked to secondary hydroxyl groups in equatorialconformation, as previously supposed from their acid lability and the IR-spectra.The glycosidic linkages of the monomers were judged from periodate oxidation,Smith degradation and permethylation studies of desulfated cell wall preparations.The predominant glycosidic linkage is 1 -+ 4. The amino sugars, galacturonicacid and the major part of glucose and galactose residues reveal this type oflinkage. Mannose and glucuronic acid are mainly 1 -+ 2 glycosidically linkedwithin the cell wall polymer. The permethylation studies clearly indicated thatthe heteroglycan is branched. The glycosidic branching points are the C-3 andC-2 hydroxyl groups of a minor part of the glucose moiety and C-3 and C-4 OH­groups of about 30 % of the galactose residues and to a lesser extent, the C-3and C-6 hydroxyl groups of some mannose residues. Glucose residues are foundat the non reducing ends of the branched structure.

On the basis of the specific rotation (en = + 48.8 0) of an aqueous solution ofthe solubilized N-acetylated cell wall it can be supposed that the majority of thesugar components are a-glycosidically linked, since the optical rotation of a­anomers of D-sugars is higher than that of p-anomers (Pazur, 1970).

More information about the linkage of the amino sugars within the cell wallpolymer was obtained from the analysis of partial hydrolysates resultingfrom the treatment with trifluoroacetic acid (Table 3). Two ninhydrin-positivealdobiuronic acids could be isolated. The glycosidic linkages of these disaccharideswere elucidated by determining the reducing end, by applying Elson-Morgan­and Morgan-Elson-tests on intact and hydrolysed disaccharides and by methyla­tion studies. Glucuronic acid is 1,4 glycosidically linked to galactosamine in disac­charide No. 1 and in disaccharide No.2 gulosaminuronic acid is 1,4-linked toglucosamine (Table 3). Disaccharide No.3 was also isolated from the partialacid hydrolysate. It is composed of glucosamine and galactose. It could be de­monstrated that galactose is present at the reducing end of the disaccharide.

From the evaluation of all the data a hypothetical structure of the brancheda-sulfated heteroglycan can be proposed (Fig. 4).

Table 3. Disaccharides isolated from partial acid hydrolysates of cell walls with trifluoro­acetic acid (6.5n trifluoroacetic acid, 100°C, 2.5 h). The disaccharides were purified bypaper chromatography (Fischer and Nebel, 1955) and paper electrophoresis at pH 1.8

Disaccharide Reducing end Non-reducing end Glycosidic linkage

1 Galactosamine Glucuronic acid Glucuronic acid-1,4-Galactosamine2 Glucosamine Gulosaminuronic Gulosaminuronic acid-1,4-Glucosamine

acid3 Galactose Glucosamine Glucosamine -J> Galactose

Chemical Composition and Structure of the Cell Wall of H. morrhuae 177

Domain I

Domain II

Domain I

y < x < n

x

Fig. 4. Proposal for the linkange of the sugar residues within the cell wall of H. morrhuaeCCM 859.Gal, galactose; GalNac, N-Acetylgalactosamine; Glc, glucose; GlcNac, N-Acetylgluco­samine; Gly, glycine; GuINUA, N-aetylgulosaminuronic acid; Man, mannose; UA, uronicacid.

Based on extraction and fragmentation studies one can distinguish at least twodomains within the heteroglycan (Fig. 4). Domain I primarily consists of uronicacids, galactosamine, mannose and glucose. Various oligosaccharides from thisdomain are found in partial acid hydrolysates of cell walls. Domain II primarilyconsists of uronic acids, glucosamine, gulosaminuronic acid and galactose andcannot be extracted as easily as components of domain I. The two domains areconnected by covalent linkages. Galactose residues of domain II constitute a pos­sible branching point. However, a part of the glycan chains may be covalentlylinked by glycine residues. At least some of the glucosamine residues are N­glycyl substituted. Dinitrophenylation and hydrazinolysis of cell walls indicatedthat the predominant portion of glycine residues is covalently linked by bothfunctional groups to cell wall components (Steber and Schleifer, 1979). Glycylpeptide bridges may exist between glucosamine and uranosyl residues of theglycan strands. Thus, at least part of the structure of cell wall of H. morrhuaemay be similar to that of bacterial peptidoglycan or pseudomurein of methano­bacteria, in that glycan strands are cross-linked through peptide linkages.

178 K.H.Schleifer, J.Steber, and H.Mayer

References

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Brown, A D., Cho, K. J.: The walls of the extremely halophilic cocci. Gram-positivebacteria lacking muramic acid. J. gen. Microbiol. 62, 267-270 (1970)

Fischer, F. G., Nebel, H. J.: Nachweis und Bestimmung von Glucosamin und Galactosaminauf Papierchromatogrammen. Hoppe Seylers Z. physiol. Chemie 302, 10-19 (1955)

Hakomori, S.: A rapid permethylation of glycolipid and polysaccharide catalysed bymethyl sulfinyl carbanion in dimethyl sulfoxide. J. Biochem. 55, 205-213 (1964)

Kocur, M., Smid, B., Martinec, T.: The fine structure of extreme halophilic cocci. Micro­bios 5, 101-107 (1972)

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Mirelman, D., Lotan, R., Bernstein, Y., Flowers, H. M., Sharon, N.: Purification andproperties of an extracellular polysaccharide containing amino sugars formed byBacillus cereus. J.gen. Microbiol. 77, 5-10 (1973)

Pazur, ].H.: Oligosaccharides. In: The Carbohydrates, Vol. II A, (W. Pigman, D. Horton,eds.). New York-London, Academic Press 1970

Reistad, R.: Cell wall of an extremely halophilic coccus. Investigation of ninhydrin­positive compounds. Arch Mikrobiol. 82, 24-30 (1972)

Reistad, R.: 2-Amino-2-dwxyguluronic acid: a constituent of the cell wall of Halococcussp., strain 24. Carbohydrate Res. 36, 420-423 (1974)

Roy, A B., Trudinger, P. A: The biochemistry of inorganic compounds of sulphur, pp.88-90. London, Cambridge University Press 1970

Steber, J., Schleifer, K. H.: Halococcus morrhuae: A sulfated heteropolysaccharide asthe structural component of the bacterial cell wall. Arch. Microbiol. 105, 173-177(1975)

Steber, J., Schleifer, K. H.: N-Glycyl-glucosamine, a novel constituent in the cell wall ofHalococeus morrhuae. Arch. Microbiol. 123, 209-212 (1979)

Professor Dr. Karl Heinz Schleifer, Lehrstuhl fur Mikrobiologie, Techn. Universitat,Arcisstr. 21, D-8000 Miinchen 2