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Typing of halophilic Archaea and characterization of their cell surface carbohydrates by use of lectins
FEMS Microbiology Letters 163 (1998) 91^97
Typing of halophilic Archaea and characterization of their cell surface carbohydrates by use of lectins Nechama Gilboa-Garber a; *, Hana Mymon a , Aharon Oren b
b
a Department of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel Division of Microbial and Molecular Ecology, The Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received 20 March 1998; accepted 6 April 1998
Abstract Lectins are important tools for cell typing and for the study of cell surface components. They have been widely used for the analysis of carbohydrates on the surface of many eukaryotic and prokaryotic cells, but they have not yet been exploited in the study of the halophilic Archaea (family Halobacteriaceae), because of the high salinity required for the structural integrity of these microorganisms. We have defined the salt concentration threshold high enough for survival of the Archaea, but sufficiently low for lectins to bind to them. Under these conditions we studied the interactions of a series of lectins, exhibiting different sugar specificities, with diverse halophilic Archaea. Concanavalin A was the most reactive by virtue of its glucose (and mannose) binding. The other lectins varied in their interactions. The results indicate that lectins might be useful probes for both archaeal typing and analysis of their cell surface carbohydrates. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lectin; Archaeal typing ; Sugar detection; Archaeal glycoprotein
1. Introduction The cytoplasmic membranes of halophilic Ar* Corresponding author. Tel.: +972 (3) 5318389; Fax: +972 (3) 5247346; E-mail: [email protected] Abbreviations: AGL, Aplysia gonad lectin; Con A, concanavalin A; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose ; GlcNAc, N-acetylglucosamine ; ECorL, Erythrina corallodendron lectin; Ha., Haloarcula; Hb., Halobacterium; Hf., Haloferax; Hr., Halorubrum; IdUA, iduronic acid; Met, methyl; MpA, Maclura pomifera agglutinin; Nm., Natronomonas; PA-IL and PA-IIL, Pseudomonas aeruginosa PA-I and PAII lectins; PNA, peanut agglutinin; SBA, soybean agglutinin; UA, uronic acid; UEA-I, Ulex europaeus lectin; WGA, wheat germ agglutinin
chaea, which require extremely high NaCl concentrations for growth and survival, are covered by cell envelopes, exhibiting a diversity of chemical structures [1], including surface (S) layers. These layers are generally heavily glycosylated, frequently with negatively charged saccharides [2,3]. The ¢rst structural descriptions of the envelope glycoprotein saccharides were those of Halobacterium salinarum [4^6] and Hb. halobium [7^9]. Subsequent gene cloning and sequencing [10^13] yielded more detailed information on the structure of the Hb. halobium S-layer and its biosynthesis, and a comparative study of the cell wall glycoproteins of the two above described species showed them to be indistinguishable by ¢n-
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Fig. 1. Model of the S-layer glycoprotein of Haloferax volcanii (A) and Halobacterium salinarum (B) and the glycosylation sites of the latter according to Lechner and Wieland [13] and Sumper et al. [18].
gerprint analysis and electrophoretic behavior [7]. In view of the near identity of these strains, they were recently classi¢ed as a single species, Hb. salinarum [14]. The latest model of their S-layer glycoprotein (molecular mass about 120 kDa) describes it as a core protein (about 87 kDa), rich in acidic amino acids, containing many acidic and neutral saccharide chains attached to it in a manner resembling animal proteoglycans [13]. It bears three types of complex saccharides (based on the composition and mode of linkage to the polypeptide chain); one of them (A) as a single copy, and the two others (B and C) in many copies, attached to three main domains along the protein core [1,10,11,13] (Fig. 1): (A) The single saccharide is a major (around 10 kDa) acidic glycosaminoglycan composed of 10^15 repeats of branched sulfated (2 SO23 4 ) pentasaccharide, containing galacturonic acid. It is bound to the sub NH2 -terminal asparagine via a special direct asparaginyl GalNAc N-linkage [8,9]. This saccharide complex is considered as the main shape-maintaining component of the S-layer. When its synthesis is in-
hibited by the antibiotic bacitracin, the rod-shaped cells are converted to spheres [2,3,13]. (B) Low molecular mass tetrasaccharide units (about 10 separate copies per molecule), composed of 2^3 sulfated (3 SO23 4 ) hexuronic acid residues (GlcUA, IdUA), attached to a glucose residue, bound by a direct N-linkage to asparagine [13,15]. (C) Collagen-like O-linked glucosyl K-1-2-galactoside disaccharide units (about 12^15 per molecule), bound via threonines, which are clustered on the protein core domain 755^774 on top of the cell membrane, close to the postulated transmembranal COO3 terminal domain. The discovery of new types of halophilic Archaea during the last two decades has shown that the family Halobacteriaceae is morphologically, physiologically, and phylogenetically diverse [16,17]. The recognition of new genera (e.g. Haloferax, Haloarcula, Halorubrum and others), whose properties di¡er greatly from Hb. salinarum, introduced the need for their typing and for a comparative study of their cell wall glycoproteins. It has been shown that Haloferax
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volcanii, like Hb. salinarum (Fig. 1), possesses glucosyl-(1-2)-galactose disaccharides O-linked to threonine residues, but the primary structures of these two proteins and their N-glycosylation patterns are distinctly di¡erent [18]. The S-layer protein of Hf. volcanii has seven N-glycosylation sites, vs. 12 in Hb. salinarum (including the sub NH2 -terminal, negatively charged repeating unit saccharide which is absent in Hf. volcanii). The N-linked oligosaccharides of Hf. volcanii are L-1-4-linked repeating glucose residues attached via asparaginyl-glucose linkages [19]. It has been suggested that the di¡erence in charge density between the extremely halophilic Hb. salinarum and Hf. volcanii, a species designated a moderate halophile with high magnesium tolerance [20], might be related to their di¡erence in salt requirement. Additional variations in the amount and nature of glycoside moieties exposed on the diverse archaeal cell surfaces may be due to either di¡erences in the structure of the S-layer glycoproteins, or the occurrence of exopolysaccharides, such as documented in Hf. mediterranei and other Archaea [21^ 24]. The aim of the present study was to introduce the application of lectins, which are highly useful and widely used for both eukaryotic and prokaryotic cell typing and analyses of their saccharides to archaeal typing and to their cell surface research. We have overcome the main obstacle of the high salinity required for the maintenance of cell integrity by examination of a series of decreasing salt concentrations in order to de¢ne the conditions enabling lectin interaction with integral archaeal cells. Under these conditions we have shown that the lectins may be used as probes for archaeal typing and for detection of cell surface carbohydrates of these prokaryotes.
2. Materials and methods 2.1. Archaeal strains The following strains were used in this study: Halobacterium salinarum (`halobium') R1, Hb. salinarum strain 5, Hf. volcanii ATCC 29605T , Hf. mediterranei ATCC 35300T , Haloarcula marismortui ATCC 43049T , Ha. vallismortis ATCC 29715T ,
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Halorubrum sodomense ATCC 33755T , and Natronomonas pharaonis strain SP1 (NCIMB 2191). 2.2. Culture conditions Cultures (1-l portions in 2-l Erlenmeyer £asks) were grown in a rotatory shaker at 35³C. The medium for growth of Hb. salinarum strains contained (all concentrations in g l31 ): NaCl, 250; KCl, 5; MgCl2 W6H2 0, 5; NH4 Cl, 5; and yeast extract, 10. Haloferax strains were grown in medium containing NaCl, 175; MgCl2 W6H2 O, 20; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; and yeast extract, 5. The Hr. sodomense medium contained: NaCl, 125; MgCl2 W6H2 O, 160; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; yeast extract, 1; casamino acids, 1; and starch, 2. The medium for Haloarcula was composed of: NaCl, 206; MgSO4 W7H2 O, 36; KCl, 0.37; CaCl2 W2H2 O, 0.5; MnCl2 , 0.013; and yeast extract, 5. All above media were adjusted to pH 7.0 with NaOH. Natronomonas was grown in medium containing NaCl, 234; Na2 CO3 , 18.5; tri-Na-citrate, 3; KCl, 2; casamino acids, 7.5; and yeast extract, 10, at pH 9.5. Cells were harvested in the late exponential phase by centrifugation (10 min, 20³C, 4200Ug). Where indicated, bacitracin was added to the media to a ¢nal concentration of 15 mg l31 . 2.3. Analysis of lectin adsorption to the archaeal cells The adsorption of the lectins to the cells was examined after 30 min incubation of 0.1 ml of the lectin solution in 0.85% NaCl solution (exhibiting a hemagglutinating titer of 1:64) with 0.2 ml of 25% (wet w/v) washed archaeal suspension in 23% NaCl solution, containing 0.1 M Tris-HCl bu¡er at pH 7.4. The supernatant £uid obtained after centrifugation (containing the residual lectin activity) was subjected to a series of 5 twofold dilutions in 0.05 ml volume with isotonic saline. Its hemagglutinating activity was compared to the original lectin titer (measured by the same series of dilutions of the unadsorbed lectin solution in 16% NaCl) after addition of 0.05 ml of 5% human erythrocyte suspension to each tube. The following lectin preparations were used: concanavalin A (Con A), Ulex europaeus lectin (UEA-I), Maclura pomifera agglutinin (MPA), peanut agglutinin (PNA), soybean agglutinin (SBA),
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Erythrina corallodendron lectin (ECorL), and wheat germ agglutinin (WGA) (all of them purchased from Sigma), as well as Pseudomonas aeruginosa PA-I and PA-II lectins (PA-IL and PA-IIL, respectively) and Aplysia gonad lectin (AGL), produced in our laboratory as previously described [25,26]. Human erythrocytes were prepared and hemagglutination tests were performed as described [26]. Papain-treated erythrocytes were used for most of the lectins, except MPL and PNA, for which sialidase-treated erythrocytes were used. The lectin adsorption onto the Archaea was graded from 0 (no adsorption) to 100% (full lectin adsorption).
3. Results Following a preliminary screening of increasing NaCl concentrations (from 0.15 to 3.0 M) which would enable lectin binding to human erythrocytes, as well as decreasing salt concentrations (from 4.5 M to 0.3 M) which would still maintain the integrity of the archaeal cells during the experiment, we de¢ned the intermediate salt concentration of 16% (= 2.7 M) as optimal to allow examination of the lectin adsorption to the archaeal cells. Con A, which binds to mannose, glucose, GlcNAc, and weakly also to glucuronic acid, inter-
Fig. 2. Adsorption of Con A to the halophilic Archaea examined, followed by the hemagglutination inhibition test. The data are presented as means þ S.E.M. obtained in 9-15 tests. The numbers representing the archaea are: (I) Halobacterium salinarum R1 (`halobium'), (II) Halobacterium salinarum strain 5 (`salinarium'), (III) Haloferax volcanii, (IV) Haloferax mediterranei, (V) Haloarcula marismortui, (VI) Haloarcula vallismortis, (VII) Halorubrum sodomense and (VIII) Natronomonas pharaonis.
Fig. 3. Comparison of the lectin adsorption onto the two Halobacterium salinarum strains: R1 (`halobium') and 5 (`salinarium'), represented by the hatched and dotted bars, respectively.
acted with all the Archaea examined (Fig. 2). Its binding to the two Hb. salinarum strains was the strongest, while its reactions with all the other archaeal species examined varied. Bacitracin treatment of Hb. salinarum R1 cells did not signi¢cantly reduce the adsorption of Con A as compared to the untreated cells. WGA, which is speci¢c to chitobiosecontaining molecules, exhibited weaker adsorption onto most Archaea examined (Figs. 3^5), but not at all to Natronomonas. The ¢ve galactophilic lectins (ECorL, MPA, PA-IL, PNA and SBA) exhibiting preferential a¤nity to galactose or GalNAc and related derivatives also exhibited lower a¤nity to these microorganisms (Figs. 3^5). The L-fucose-speci¢c UEA-I lectin and the L-fucose- and D-mannose-speci¢c lectin PA-IIL exhibited moderate adsorption onto these Archaea (Figs. 3^5). The most dramatic results were obtained with the Aplysia lectin AGL, which exhibits the highest a¤n-
Fig. 4. Comparison of the lectin adsorption onto Haloferax volcanii and Haloferax mediterranei (hatched and dotted bars, respectively).
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Fig. 5. Comparison of the lectin adsorption onto Haloarcula marismortui and Halolarcula vallismortis (hatched and dotted bars, respectively).
ity for galacturonic acid, but also reacts (with much lower a¤nity) with D-galactose. As may be seen in Figs. 3^6, this lectin very strongly adsorbed onto the two Hb. salinarum strains: R1 (`halobium') and 5 (`salinarium'), and onto Ha. vallismortis. In contrast, the activity of this lectin was not reduced by Hf. volcanii, and only very weakly by Hf. mediterranei (Fig. 6).
4. Discussion Lectins, which are ubiquitous selective sugar binding proteins, are widely used for the detection of cell surface carbohydrates and cell typing in both prokaryotic and eukaryotic organisms. Owing to their antibody-like function, combined with inhibitability by simple carbohydrates, they are advantageous probes for terminal carbohydrate analyses
Fig. 6. Reduction of AGL hemagglutination titer following adsorption onto the halophilic Archaea examined (for details see Fig. 2).
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[27,28]. So far, they have not been used for the analysis of halophilic Archaea due to the high salinity required for survival of these organisms. Such salinity abolishes both antibody and lectin interactions. We have identi¢ed a threshold intermediate salt concentration, supporting both the halophilic archaeal integrity and lectin binding to them. Under these conditions the adsorption of nine lectins (exhibiting sugar speci¢cities for glucose, galactose, and their derivatives, mannose, fucose, and galacturonic acid) onto eight halophilic Archaea from ¢ve genera was examined, using the hemagglutination inhibition test. Con A, which detects mannose and glucose (and the derivatives GlcNAc and glucuronic acid), reacted, as anticipated, with all the Archaea examined (Fig. 2). Its very strong adsorption onto both Hb. salinarum strains and Hf. volcanii (Fig. 2) nicely accorded with the reports on presence of terminal glucose in the O-threonine-linked Glc1-2 Gal disaccharide clusters at the cell membrane proximity and of glucuronic acid in the N-asparagine-linked tetrasaccharides of the ¢rst two strains [13,18], and in the Nasparagine linked decasaccharides Glc-(L1-4 Glc)8 L1-4 Glc-Asn of Hf. volcanii [19] (Fig. 1). The fact that the bacitracin-treated Hb. salinarum cells strongly adsorbed Con A, despite the proposed absence of the sulfated repeating unit saccharide following this treatment [7,10], also ¢ts with the suggestion that Con A only reacts with the short saccharides of this bacterium. The sulfate anions, which are considered important for the high salt adaptation of the more extremely halophilic Archaea, and probably also protect the underlying sugars from degradation by masking [29], may reduce lectin adsorption. Removal of such anions [30] sometimes enables additional lectin adsorption, as was shown with the highly sulfated adhesive proteoglycan of the marine sponge Microciona prolifera [31]. There it was reported that desulfation greatly enhanced the proteoglycan interaction with Con A, WGA, PNA and UEA-I. However, our attempt to use their desulfation method with methanol-HCl at 62.5³C for 4 h [31] did not signi¢cantly increase the adsorbance of these lectins to the Archaea. It is interesting to note that Con A also interacted with chondroitin sulfate, which exhibits structural similarity to some archaeal heteropolysaccharides [13].
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The results presented in Fig. 3 show that in contrast to Con A, the interactions of the (GlcNAc)2 speci¢c WGA and the galactophilic lectins SBA, PNA and PA-I with the Halobacterium strains were relatively weak, whereas AGL (which is outstanding in its much higher a¤nity to galacturonic acid [25]) adsorbed very strongly onto the two Halobacterium strains, whilst exhibiting insigni¢cant adsorption onto the two Haloferax strains (Figs. 3^6). This profound AGL di¡erentiation between the Halobacterium and Haloferax strains agrees nicely with the reports of Sumper et al. [18,19], who cloned and sequenced the Hf. volcanii gene encoding the S-layer glycoprotein, and have shown that Hf. volcanii lacks the galacturonic acid-containing glycosaminoglycan linked to the sub NH2 -terminal asparagine. The reported structure of the mucoid envelope of this Archaeon [23], which is a highly charged sulfated heteropolysaccharide composed of four GlcNAcUA 1-6 Man 1-4 GlcNAc(3-SO23 4 )UA1 repeating units [24] without galacturonic acid residues, explains the negative result with AGL. The data presented in Figs. 5 and 6 demonstrate that although both Haloarcula species (Ha. marismortui and Ha. vallismortis) exhibit moderate Con A adsorption, they di¡er signi¢cantly in interactions with AGL: while Ha. marismortui exhibited moderate AGL binding, Ha. vallismortis resembled Hb. salinarum strains in strong adsorption of this lectin, indicating either high galacturonic acid concentration or acidity. Another signi¢cant di¡erence between these two Haloarcula species is the stronger adsorption of the fucose binding lectins PA-IIL and UEA-I to Ha. marismortui as compared to Ha. vallismortis, which is probably poor in, or lacks, terminal fucose. As seen in Fig. 6, AGL adsorption onto the two additional Archaea examined, Hr. sodomense and Nm. pharaonis, was very similar, considerably lower than that observed with Hb. salinarum strains and Ha. vallismortis. The adsorption of WGA and the galactophilic lectins onto these two strains was insigni¢cant. UEA-I exhibited a low degree of adsorption to Hr. sodomense, but not to Nm. pharaonis. The above results indicate that under de¢ned conditions, lectins may be used for typing, identi¢cation, and characterization of the cell wall carbohydrates of the halophilic Archaea.
Acknowledgments The work was supported by the Bar-Ilan University Research Foundation. We thank Avrille Goldreich, Mrs. Sharon Victor and Mrs. Ella Gindi for the skillful typing of the manuscript and the graphic presentation.
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N. Gilboa-Garber et al. / FEMS Microbiology Letters 163 (1998) 91^97 [16] Tindall, B.J. (1992) The family Halobacteriaceae. In: The Prokaryotes. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identi¢cation, Applications (Balows, A., Truëper, H.G., Dworkin, M., Harder, W. and Schleifer, K.H., Eds.), Vol. I, pp. 768^808. Springer-Verlag, New York. [17] Torreblanca, M., Rodriguez-Valera, F., Juez, G., Ventosa, A., Kamekura, M. and Kates, M. (1986) Classi¢cation of nonalkaliphilic halobacteria based on numerical taxonomy and polar lipid composition, and description of Haloarcula gen. nov., and Haloferax gen. nov. Syst. Appl. Microbiol. 8, 89^ 99. [18] Sumper, M., Berg, E., Mengele, R. and Strobel, I. (1990) Primary structure and glycosylation of the S-layer protein of Haloferax volcanii. J. Bacteriol. 172, 7111^7118. [19] Mengele, R. and Sumper, M. (1992) Drastic di¡erences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles. J. Biol. Chem. 267, 8182^8185. [20] Mullakhanbhai, M.F. and Larsen, H. (1975) Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch. Microbiol. 104, 207^214. [21] Steber, J. and Schleifer, K.H. (1975) Halococcus morrhuae: A sulfated heteropolysaccharide as the structural component of the bacterial cell wall. Arch. Microbiol. 105, 173^177. [22] Schleifer, K.-H., Steber, J. and Mayer, H. (1982) Chemical composition and structure of the cell wall of Halococcus morrhuae. Zbl. Bakt. Hyg. I. Abt. Orig. C 3, 171^178. [23] Anton, J., Meseguer, I. and Rodriguez-Valera, F. (1988) Production of an extracellular polysaccharide by Haloferax mediterranei. Appl. Environ. Microbiol. 54, 2381^2386. [24] Parolis, H., Parolis, L.A.S., Boan, I.F., Rodriguez-Valera, F., Widmalm, G., Manca, M.C., Jansson, P.E. and Sutherland,
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Typing of halophilic Archaea and characterization of their cell surface carbohydrates by use of lectins Nechama Gilboa-Garber a; *, Hana Mymon a , Aharon Oren b
b
a Department of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel Division of Microbial and Molecular Ecology, The Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received 20 March 1998; accepted 6 April 1998
Abstract Lectins are important tools for cell typing and for the study of cell surface components. They have been widely used for the analysis of carbohydrates on the surface of many eukaryotic and prokaryotic cells, but they have not yet been exploited in the study of the halophilic Archaea (family Halobacteriaceae), because of the high salinity required for the structural integrity of these microorganisms. We have defined the salt concentration threshold high enough for survival of the Archaea, but sufficiently low for lectins to bind to them. Under these conditions we studied the interactions of a series of lectins, exhibiting different sugar specificities, with diverse halophilic Archaea. Concanavalin A was the most reactive by virtue of its glucose (and mannose) binding. The other lectins varied in their interactions. The results indicate that lectins might be useful probes for both archaeal typing and analysis of their cell surface carbohydrates. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lectin; Archaeal typing ; Sugar detection; Archaeal glycoprotein
1. Introduction The cytoplasmic membranes of halophilic Ar* Corresponding author. Tel.: +972 (3) 5318389; Fax: +972 (3) 5247346; E-mail: [email protected] Abbreviations: AGL, Aplysia gonad lectin; Con A, concanavalin A; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose ; GlcNAc, N-acetylglucosamine ; ECorL, Erythrina corallodendron lectin; Ha., Haloarcula; Hb., Halobacterium; Hf., Haloferax; Hr., Halorubrum; IdUA, iduronic acid; Met, methyl; MpA, Maclura pomifera agglutinin; Nm., Natronomonas; PA-IL and PA-IIL, Pseudomonas aeruginosa PA-I and PAII lectins; PNA, peanut agglutinin; SBA, soybean agglutinin; UA, uronic acid; UEA-I, Ulex europaeus lectin; WGA, wheat germ agglutinin
chaea, which require extremely high NaCl concentrations for growth and survival, are covered by cell envelopes, exhibiting a diversity of chemical structures [1], including surface (S) layers. These layers are generally heavily glycosylated, frequently with negatively charged saccharides [2,3]. The ¢rst structural descriptions of the envelope glycoprotein saccharides were those of Halobacterium salinarum [4^6] and Hb. halobium [7^9]. Subsequent gene cloning and sequencing [10^13] yielded more detailed information on the structure of the Hb. halobium S-layer and its biosynthesis, and a comparative study of the cell wall glycoproteins of the two above described species showed them to be indistinguishable by ¢n-
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Fig. 1. Model of the S-layer glycoprotein of Haloferax volcanii (A) and Halobacterium salinarum (B) and the glycosylation sites of the latter according to Lechner and Wieland [13] and Sumper et al. [18].
gerprint analysis and electrophoretic behavior [7]. In view of the near identity of these strains, they were recently classi¢ed as a single species, Hb. salinarum [14]. The latest model of their S-layer glycoprotein (molecular mass about 120 kDa) describes it as a core protein (about 87 kDa), rich in acidic amino acids, containing many acidic and neutral saccharide chains attached to it in a manner resembling animal proteoglycans [13]. It bears three types of complex saccharides (based on the composition and mode of linkage to the polypeptide chain); one of them (A) as a single copy, and the two others (B and C) in many copies, attached to three main domains along the protein core [1,10,11,13] (Fig. 1): (A) The single saccharide is a major (around 10 kDa) acidic glycosaminoglycan composed of 10^15 repeats of branched sulfated (2 SO23 4 ) pentasaccharide, containing galacturonic acid. It is bound to the sub NH2 -terminal asparagine via a special direct asparaginyl GalNAc N-linkage [8,9]. This saccharide complex is considered as the main shape-maintaining component of the S-layer. When its synthesis is in-
hibited by the antibiotic bacitracin, the rod-shaped cells are converted to spheres [2,3,13]. (B) Low molecular mass tetrasaccharide units (about 10 separate copies per molecule), composed of 2^3 sulfated (3 SO23 4 ) hexuronic acid residues (GlcUA, IdUA), attached to a glucose residue, bound by a direct N-linkage to asparagine [13,15]. (C) Collagen-like O-linked glucosyl K-1-2-galactoside disaccharide units (about 12^15 per molecule), bound via threonines, which are clustered on the protein core domain 755^774 on top of the cell membrane, close to the postulated transmembranal COO3 terminal domain. The discovery of new types of halophilic Archaea during the last two decades has shown that the family Halobacteriaceae is morphologically, physiologically, and phylogenetically diverse [16,17]. The recognition of new genera (e.g. Haloferax, Haloarcula, Halorubrum and others), whose properties di¡er greatly from Hb. salinarum, introduced the need for their typing and for a comparative study of their cell wall glycoproteins. It has been shown that Haloferax
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volcanii, like Hb. salinarum (Fig. 1), possesses glucosyl-(1-2)-galactose disaccharides O-linked to threonine residues, but the primary structures of these two proteins and their N-glycosylation patterns are distinctly di¡erent [18]. The S-layer protein of Hf. volcanii has seven N-glycosylation sites, vs. 12 in Hb. salinarum (including the sub NH2 -terminal, negatively charged repeating unit saccharide which is absent in Hf. volcanii). The N-linked oligosaccharides of Hf. volcanii are L-1-4-linked repeating glucose residues attached via asparaginyl-glucose linkages [19]. It has been suggested that the di¡erence in charge density between the extremely halophilic Hb. salinarum and Hf. volcanii, a species designated a moderate halophile with high magnesium tolerance [20], might be related to their di¡erence in salt requirement. Additional variations in the amount and nature of glycoside moieties exposed on the diverse archaeal cell surfaces may be due to either di¡erences in the structure of the S-layer glycoproteins, or the occurrence of exopolysaccharides, such as documented in Hf. mediterranei and other Archaea [21^ 24]. The aim of the present study was to introduce the application of lectins, which are highly useful and widely used for both eukaryotic and prokaryotic cell typing and analyses of their saccharides to archaeal typing and to their cell surface research. We have overcome the main obstacle of the high salinity required for the maintenance of cell integrity by examination of a series of decreasing salt concentrations in order to de¢ne the conditions enabling lectin interaction with integral archaeal cells. Under these conditions we have shown that the lectins may be used as probes for archaeal typing and for detection of cell surface carbohydrates of these prokaryotes.
2. Materials and methods 2.1. Archaeal strains The following strains were used in this study: Halobacterium salinarum (`halobium') R1, Hb. salinarum strain 5, Hf. volcanii ATCC 29605T , Hf. mediterranei ATCC 35300T , Haloarcula marismortui ATCC 43049T , Ha. vallismortis ATCC 29715T ,
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Halorubrum sodomense ATCC 33755T , and Natronomonas pharaonis strain SP1 (NCIMB 2191). 2.2. Culture conditions Cultures (1-l portions in 2-l Erlenmeyer £asks) were grown in a rotatory shaker at 35³C. The medium for growth of Hb. salinarum strains contained (all concentrations in g l31 ): NaCl, 250; KCl, 5; MgCl2 W6H2 0, 5; NH4 Cl, 5; and yeast extract, 10. Haloferax strains were grown in medium containing NaCl, 175; MgCl2 W6H2 O, 20; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; and yeast extract, 5. The Hr. sodomense medium contained: NaCl, 125; MgCl2 W6H2 O, 160; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; yeast extract, 1; casamino acids, 1; and starch, 2. The medium for Haloarcula was composed of: NaCl, 206; MgSO4 W7H2 O, 36; KCl, 0.37; CaCl2 W2H2 O, 0.5; MnCl2 , 0.013; and yeast extract, 5. All above media were adjusted to pH 7.0 with NaOH. Natronomonas was grown in medium containing NaCl, 234; Na2 CO3 , 18.5; tri-Na-citrate, 3; KCl, 2; casamino acids, 7.5; and yeast extract, 10, at pH 9.5. Cells were harvested in the late exponential phase by centrifugation (10 min, 20³C, 4200Ug). Where indicated, bacitracin was added to the media to a ¢nal concentration of 15 mg l31 . 2.3. Analysis of lectin adsorption to the archaeal cells The adsorption of the lectins to the cells was examined after 30 min incubation of 0.1 ml of the lectin solution in 0.85% NaCl solution (exhibiting a hemagglutinating titer of 1:64) with 0.2 ml of 25% (wet w/v) washed archaeal suspension in 23% NaCl solution, containing 0.1 M Tris-HCl bu¡er at pH 7.4. The supernatant £uid obtained after centrifugation (containing the residual lectin activity) was subjected to a series of 5 twofold dilutions in 0.05 ml volume with isotonic saline. Its hemagglutinating activity was compared to the original lectin titer (measured by the same series of dilutions of the unadsorbed lectin solution in 16% NaCl) after addition of 0.05 ml of 5% human erythrocyte suspension to each tube. The following lectin preparations were used: concanavalin A (Con A), Ulex europaeus lectin (UEA-I), Maclura pomifera agglutinin (MPA), peanut agglutinin (PNA), soybean agglutinin (SBA),
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Erythrina corallodendron lectin (ECorL), and wheat germ agglutinin (WGA) (all of them purchased from Sigma), as well as Pseudomonas aeruginosa PA-I and PA-II lectins (PA-IL and PA-IIL, respectively) and Aplysia gonad lectin (AGL), produced in our laboratory as previously described [25,26]. Human erythrocytes were prepared and hemagglutination tests were performed as described [26]. Papain-treated erythrocytes were used for most of the lectins, except MPL and PNA, for which sialidase-treated erythrocytes were used. The lectin adsorption onto the Archaea was graded from 0 (no adsorption) to 100% (full lectin adsorption).
3. Results Following a preliminary screening of increasing NaCl concentrations (from 0.15 to 3.0 M) which would enable lectin binding to human erythrocytes, as well as decreasing salt concentrations (from 4.5 M to 0.3 M) which would still maintain the integrity of the archaeal cells during the experiment, we de¢ned the intermediate salt concentration of 16% (= 2.7 M) as optimal to allow examination of the lectin adsorption to the archaeal cells. Con A, which binds to mannose, glucose, GlcNAc, and weakly also to glucuronic acid, inter-
Fig. 2. Adsorption of Con A to the halophilic Archaea examined, followed by the hemagglutination inhibition test. The data are presented as means þ S.E.M. obtained in 9-15 tests. The numbers representing the archaea are: (I) Halobacterium salinarum R1 (`halobium'), (II) Halobacterium salinarum strain 5 (`salinarium'), (III) Haloferax volcanii, (IV) Haloferax mediterranei, (V) Haloarcula marismortui, (VI) Haloarcula vallismortis, (VII) Halorubrum sodomense and (VIII) Natronomonas pharaonis.
Fig. 3. Comparison of the lectin adsorption onto the two Halobacterium salinarum strains: R1 (`halobium') and 5 (`salinarium'), represented by the hatched and dotted bars, respectively.
acted with all the Archaea examined (Fig. 2). Its binding to the two Hb. salinarum strains was the strongest, while its reactions with all the other archaeal species examined varied. Bacitracin treatment of Hb. salinarum R1 cells did not signi¢cantly reduce the adsorption of Con A as compared to the untreated cells. WGA, which is speci¢c to chitobiosecontaining molecules, exhibited weaker adsorption onto most Archaea examined (Figs. 3^5), but not at all to Natronomonas. The ¢ve galactophilic lectins (ECorL, MPA, PA-IL, PNA and SBA) exhibiting preferential a¤nity to galactose or GalNAc and related derivatives also exhibited lower a¤nity to these microorganisms (Figs. 3^5). The L-fucose-speci¢c UEA-I lectin and the L-fucose- and D-mannose-speci¢c lectin PA-IIL exhibited moderate adsorption onto these Archaea (Figs. 3^5). The most dramatic results were obtained with the Aplysia lectin AGL, which exhibits the highest a¤n-
Fig. 4. Comparison of the lectin adsorption onto Haloferax volcanii and Haloferax mediterranei (hatched and dotted bars, respectively).
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Fig. 5. Comparison of the lectin adsorption onto Haloarcula marismortui and Halolarcula vallismortis (hatched and dotted bars, respectively).
ity for galacturonic acid, but also reacts (with much lower a¤nity) with D-galactose. As may be seen in Figs. 3^6, this lectin very strongly adsorbed onto the two Hb. salinarum strains: R1 (`halobium') and 5 (`salinarium'), and onto Ha. vallismortis. In contrast, the activity of this lectin was not reduced by Hf. volcanii, and only very weakly by Hf. mediterranei (Fig. 6).
4. Discussion Lectins, which are ubiquitous selective sugar binding proteins, are widely used for the detection of cell surface carbohydrates and cell typing in both prokaryotic and eukaryotic organisms. Owing to their antibody-like function, combined with inhibitability by simple carbohydrates, they are advantageous probes for terminal carbohydrate analyses
Fig. 6. Reduction of AGL hemagglutination titer following adsorption onto the halophilic Archaea examined (for details see Fig. 2).
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[27,28]. So far, they have not been used for the analysis of halophilic Archaea due to the high salinity required for survival of these organisms. Such salinity abolishes both antibody and lectin interactions. We have identi¢ed a threshold intermediate salt concentration, supporting both the halophilic archaeal integrity and lectin binding to them. Under these conditions the adsorption of nine lectins (exhibiting sugar speci¢cities for glucose, galactose, and their derivatives, mannose, fucose, and galacturonic acid) onto eight halophilic Archaea from ¢ve genera was examined, using the hemagglutination inhibition test. Con A, which detects mannose and glucose (and the derivatives GlcNAc and glucuronic acid), reacted, as anticipated, with all the Archaea examined (Fig. 2). Its very strong adsorption onto both Hb. salinarum strains and Hf. volcanii (Fig. 2) nicely accorded with the reports on presence of terminal glucose in the O-threonine-linked Glc1-2 Gal disaccharide clusters at the cell membrane proximity and of glucuronic acid in the N-asparagine-linked tetrasaccharides of the ¢rst two strains [13,18], and in the Nasparagine linked decasaccharides Glc-(L1-4 Glc)8 L1-4 Glc-Asn of Hf. volcanii [19] (Fig. 1). The fact that the bacitracin-treated Hb. salinarum cells strongly adsorbed Con A, despite the proposed absence of the sulfated repeating unit saccharide following this treatment [7,10], also ¢ts with the suggestion that Con A only reacts with the short saccharides of this bacterium. The sulfate anions, which are considered important for the high salt adaptation of the more extremely halophilic Archaea, and probably also protect the underlying sugars from degradation by masking [29], may reduce lectin adsorption. Removal of such anions [30] sometimes enables additional lectin adsorption, as was shown with the highly sulfated adhesive proteoglycan of the marine sponge Microciona prolifera [31]. There it was reported that desulfation greatly enhanced the proteoglycan interaction with Con A, WGA, PNA and UEA-I. However, our attempt to use their desulfation method with methanol-HCl at 62.5³C for 4 h [31] did not signi¢cantly increase the adsorbance of these lectins to the Archaea. It is interesting to note that Con A also interacted with chondroitin sulfate, which exhibits structural similarity to some archaeal heteropolysaccharides [13].
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The results presented in Fig. 3 show that in contrast to Con A, the interactions of the (GlcNAc)2 speci¢c WGA and the galactophilic lectins SBA, PNA and PA-I with the Halobacterium strains were relatively weak, whereas AGL (which is outstanding in its much higher a¤nity to galacturonic acid [25]) adsorbed very strongly onto the two Halobacterium strains, whilst exhibiting insigni¢cant adsorption onto the two Haloferax strains (Figs. 3^6). This profound AGL di¡erentiation between the Halobacterium and Haloferax strains agrees nicely with the reports of Sumper et al. [18,19], who cloned and sequenced the Hf. volcanii gene encoding the S-layer glycoprotein, and have shown that Hf. volcanii lacks the galacturonic acid-containing glycosaminoglycan linked to the sub NH2 -terminal asparagine. The reported structure of the mucoid envelope of this Archaeon [23], which is a highly charged sulfated heteropolysaccharide composed of four GlcNAcUA 1-6 Man 1-4 GlcNAc(3-SO23 4 )UA1 repeating units [24] without galacturonic acid residues, explains the negative result with AGL. The data presented in Figs. 5 and 6 demonstrate that although both Haloarcula species (Ha. marismortui and Ha. vallismortis) exhibit moderate Con A adsorption, they di¡er signi¢cantly in interactions with AGL: while Ha. marismortui exhibited moderate AGL binding, Ha. vallismortis resembled Hb. salinarum strains in strong adsorption of this lectin, indicating either high galacturonic acid concentration or acidity. Another signi¢cant di¡erence between these two Haloarcula species is the stronger adsorption of the fucose binding lectins PA-IIL and UEA-I to Ha. marismortui as compared to Ha. vallismortis, which is probably poor in, or lacks, terminal fucose. As seen in Fig. 6, AGL adsorption onto the two additional Archaea examined, Hr. sodomense and Nm. pharaonis, was very similar, considerably lower than that observed with Hb. salinarum strains and Ha. vallismortis. The adsorption of WGA and the galactophilic lectins onto these two strains was insigni¢cant. UEA-I exhibited a low degree of adsorption to Hr. sodomense, but not to Nm. pharaonis. The above results indicate that under de¢ned conditions, lectins may be used for typing, identi¢cation, and characterization of the cell wall carbohydrates of the halophilic Archaea.
Acknowledgments The work was supported by the Bar-Ilan University Research Foundation. We thank Avrille Goldreich, Mrs. Sharon Victor and Mrs. Ella Gindi for the skillful typing of the manuscript and the graphic presentation.
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