Molecular Organization of Selected Prokaryotic S-Iayer Proteins

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REVIEW I SYNTHESE " -

Molecular organization of selected prokaryotic S-Iayer proteins

Harald Claus, Erol Akc;a, Tony Debaerdemaeker, Christine Evrard, Jean-Paul Declercq, J. Robin Harris, Bernhard Schlott, and Helmut Konig

Abstract: Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-rprotein species with molecular masses rang ing from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture. The primary sequen ces of hyperthermophilic archaeal species exhibit some characteristic signatures, Further adaptations to their specific environments occur by various post-translational modifications, such as linkage of glycans, lipids, phosphate, and sul fate groups to the protein or by proteolytic processing. Specific domains direct the anchoring of the S-layer to the un derlying cell wall components and transport across the cytoplasma memhrane. In addition to their presumptive original role as protective coats in archaea and bacteria, they have adapted new functions, e.g., as molecular sieves, attachment sites for extracellular enzymes, and virulence factors.

Key words: prokaryotes, cell walls, S-layer (glyco-) proteins, protein stabilization.

Resume: Les couches de surface (couches S) cristallines regulieres sont repandues parmi les procaryotes et represen tent probablement les structures de parois cellulaires primitives. Des genes de couches S ont ete retrouves chez environ 400 especes. differentes des domaines procaryotes bacteria et archaea. Les couches S sont habituelJement constituees dun seul type de glycoproreine de masses moleculaires allant de 40 it 200 kDa qui forment des reseaux ayant une ar chitecture oblique, retragonale au hexagonale. Les sequences primaires des espPces acheales hyperthermophiles presen tent certaines caracteristiques distinctives. Des adaptations subsequenres it leurs environnements specifiques furent facilitees par diverses modifications post-traductionnelles telles que des liens de la proteine it des glycancs, des lipides, des groupes phosphates et sulfates ou par une transformation proteolytique. Des domaines specifiques sont responsables de l' ancrage de la couche S aux composantes de la paroi cellulaire inferieure et au transport a travers la membrane cytoplasrnique. En plus de leur role original presume comme couche protectrice des archeobacteries et des eubacteries, elles ant adopte de nouvelles fonctions. p. ex. des tarnis moleculaires, des sites d' attachernent pour des enzymes extra cellulaires et des facteurs de virulence.

Mots des : procaryotes, paroi celJulaire, glycoproteines de la couches S, stabilisation proteique.

[Traduit par Ia Redaction]

Introduction the S-layer, is regarded as the most ancient biological mem brane that is ubiquitous and maintained in most species of During evolution, prokaryotic microorganisms adapted var bacteria and archaea (Baumeister et al. 1989: Beveridge ane! ions 1'.f>11 wa]] structures in rf>sponsf> to their spf>(~ifi(' and of Koval 1992; Messner and Sleytr 1992; Murray 1993; Sleytr ten harsh habitats. A rigid proteinaceous envelope, termed et al. 1993; Sleytr 1997; Konig and Messner 1997). Mono-

Received 3 September 2004, Revision received 17 February 2005. Accepted 3 March 2005. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 21 Octoher 2005. H. Claus, Eo Akca, and H. Koni ,.t Instit.ut}lir Mikrobiologie und Weinforschung, Johannes Gutenberg-Universitat Mainz, Becherweg IS, D-55099 Mainz, Germany. T. Debaerdemaeker, Sektion Rontgen- und Elektronenbeugung, Alben Einstein-Universitat Ulrn. D-89069 Ulm, Germany. C, Evrard and .T.-P. Declercq. Unite de Chimie Structurale, Universite Catholique de Louvain. ] Place Louis Pasteur, B-1348 Louvain-Ia-Neuvc, Belgium. J,R. Harris. Institut fur Zoologic. Abt. II, Johannes Gutenberg-Universitat Mainz, MiilJerweg 6. D-55099 Mainz, Germany. B. Scblott. Institur fiir Molekulare Biotechnologic. Abt. Biochemie, Beutenbergstr. 1[, D-07745 Jenu, Germany. ICorresponding author (e-mail: hkoenigtsiuni-mainz.de).

Can. J. Microbial. 51: 731-743 (200S,I doi: 10.1139/WOS-093 @ 200S NRC Canada Can. --'. Microbial. Vol. 51. 2005

molecular arrays of proteinaceous subunits have' also been pepridogycan or by secondary cell wall polymers. e.g.. teichoic observed in archaeal sheaths (Beveridge et a1. 1990) and on acids, teichuronic acids. lipoteichoic acids. or lipoglycans the surface of the cell wall of cyanobacteria (Smarda et a!. (Egelseer et aJ. 1998; Smit et al. 2001: Antikainen et al. 2002). The S-]ayers usually consist of a single type of pro 20m). This attachment is mediated by so-called S-Iayer tein that bas the intrinsic feature of being able to assemble homologous (5LH) domains found in some Bacillaceae, into highly ordered two-dimensional crystalline structures. Deinococcus radiodurans, Thcrmotoga maritima, and the In some archaea and bacteria, two superimposed S-Iayer lat Gram-negative bacteria Synechocvstis sp. and Thennus ther tices, composed of different subunit species, may be present mophilus (Archibald et al. 1993), but not all S-layer proteins (Sleytr et a!. 2001). In most archaea, the S-layer has a hex possess SLH domains (cf. Geobacillus stearothermophilus, agonal lattice symmetry, whereas in bacteria. oblique and see Table 3). SLH domains were identified not only at the tetragonal lattices are found more frequently (Konig 1988; N-terminal part of S-layer proteins but also at the C-terminal Messner and Sleytr 1992; Sleytr et al. 1996) The centre-to part of cell-associated exoproteins and enzymes of Gram centre distance of the morphological units varies between positive bacteria (Lnpas et a!. 1994; Engelhardt and Peters 2.5 and 35 nm and the thickness of the layer between 5.0 19981. Typically, S-layer proteins and celJ-associated exo and 25 nm in bacteria and up to 70 nm in archaea. Owing to enzymes possess three repeats of SLH motifs, each consist their crystalline nature. S-Iayer' lattices contain pores of ing of 50-70 amino acids (Engelhardt and Peters 1998; identical size, usually between 2 and 8 nm, and morphology Mesnage et al. 2000; Sara and Sleytr 2000i. The N-terminal (Sleytr et a!. 2001). This strict modular construction of the sequences fol1owing the docking regions are necessary for S-Iayer lattice is the basis for many promising applications the self-assembly process as found for the S-layer proteins in nanobioteclmology iSleytr et a!. 1999. 200 I; Ilk et a!. from Bacillus and Geobocillus, whereas aoout200 amino 2002; Schaffer and Messner 2004). aeids could be truncated at the C terminus without negative Depending on the individual cell walJ composition of the effect in this respect (.Tarosch et a!. 200I: Ilk et al. 2002; microorganism. the S-layer is noncovalently attached to the Runzler et al, 2004). cytoplasmic membrane, outer membrane, peptidoglycan, pseudornurein, or secondary cell wall components, respec tively (Sleytr et a!. 2001). The S-layer proteins contain about Post-translational modifications 40-60 mol% hydrophobic residues and 25 mol% charged Proteolytic cleavage of N- and C-terminal fragments, amino acids (Sleytr 1997; Sleytr and Beveridge 1999; Sara phosphorylation, sulphurylation, glycosylation, and lipid trans and Sleytr 2000). Occurrence of cysteine is typically for the fer are well-documented mechanisms that alter sizes and mo S-]ayer proteins of hypertherrnophilic archaea, which also lecular features of translated prokaryotic S-layer proteins have more charged residues compared with their mesophilic (Boot and Pouwels 1996: Eichler 2003). By proteolytic cleav counterparts (Ak<;a et a!. 2002; Akca 2004; Claus ct al. age of a proprotein. two S-layer proteins arise in Clostridium 2002). Most S-layer proteins are weakly acidic, but some are difficile (Calabi et a!. 2001). Phosphorylation of tyrosine res basic, e.g., those of Methanothermus [ervidus, Metlianother idues has been demonstrated for the S-Iayer protein of Aem mus sociabilis, Mcthanosarcina I7IGzei, Mrthanobarrerium monos hydrophila (Thomas and Trust 1995). The S-layer thennoautotrophicum, and Lactobacilli with isoelectric points proteins of the archaeal halobacteria are modified by highly between pH 8.0 and 10.0 (Brockl et a!. J99 J: Skytr 1997; sulfonated glycans and represent one of the first prokaryotic Claus et al. 2002). The amino acids of S-layer proteins are glycoproteins ever described (Mescher and Strominger 1976; ~-sheets organized such that about 40% are as and 10% Lechner and Sumper 1987; Sumper 1993). After the first 20% are as a-helices. Their molecular masses range from 40 description of glycosylated bacterial S-Iayer proteins in to 200 kDa. thermophilic Clostridia (Sleytr and Thorne 1976), presently about 40 different S-Jayer glycan structures have been fully Domains or at least partially e1ucitated in the Gram-positive bacterial genera Bacillus, Geobacillus, Lactobacillus. and Clostridium Bacterial S-Iayer proteins are secreted by either the uni as well as in archaea (Karcher et al. 1993: Sumper and versally conserved general pathway (SEC) or an ATP-binding Wieland 1995; Eichler 2003; Upreti et al. 2003: Schaffer and cassette transporter (Fernandez and Berenguer 2000; Sara Messner 2004). Glycosylation of S-layer proteins generally and Sleytr 2000; Kawai et al. 1998). S-Iayer proteins [rom varies between 2% and 10% (m/m). Bacterial S-layer glycan Mcthanothennus spp. and Methanococcus spp. were pre chains are linear or branched homo- or hetero-saccharides, dicted to be secreted by the SEC pathway (Brockl et a!. which comprise 20-50 identical repeating units, whereas 1991; Akqa et a!. 2002), which is a common route for in archaea, short oligosaccharides prevail (Karcher et a1. archaeal protein translocation (Eichler 2000). Indeed, most 1993: Schaffer and Messner 2(04). The monosaccharide prokaryotic S-layer proteins exhibit a typical 30 amino acid constituents include a wide range of neutral hexoses, G lead@!' pept;de with a positively charged N terminus, a hy deoxyhexoses. and amino sugars. Whereas in archaea, 0 drophobic core, and a Csterminal recognition site for the and N-glycosidic linkages may exist simultaneollsly in the cleavage by specific signal peptidases (Brendtsen et a!. 2004). same strain, only one linkage scheme, predominantly 0 The S-layer proteins from some species share homologous bonds. occurs in the Gram-positive bacteria. Lipid modifica domains with specific functions. Most halobacrerial S-byer tion of S-layer (glyco-)proteins has been demonstrated for proteins are anchored in the cytoplasmic membrane by a C the halophilic archaea Haloferax volcanii and Halohacter terminal transmembrane domain. The S-layer of Gram-positive ium halobium (Eichler 2003; Konrad and Eichler 2002l. bacteria is fixed by its N terminus or C terminus to the Like glycosylation, lipid attachment takes place on the exrer

nul cell surface. i.e.. following protein translocation across contribute to the pathogenicity of these microorganisms the plasma membrane. (Carl and Dasch 1989: Etienne-Toumelin et al. 1995; Post-translational modifications and (or! intrinsic features Kotiranta et al. J997; Mesnage et al. 19(7). The S-layers of of the S-byer proteins sometimes provoke an unusual elec Lactolracillus acidophilus and Lactobacillus crispatus cells trophoretic behaviour and thus confusion about the exact (Schneitz et al. 1993) and Clostridium difficile (Calabi et al. molecular mass of the monomer when comparing results 200lJ strains mediate adhesion to mammalian gut epithelial from SDS-PAGE and gene sequences. As an example, the cells. purified S-Iayer glycoprotein from Methanothermus [ervidus S-Jayers from Bacillaceae were found to function as adhe exhibits two size conformations on SDS gels (Brock! et a!. sion sites for cell-associated exoenzyrnes, e.g., high molecu 1991). The electrophoretic migration of the purified S-layer lar mass exoamylase from Geobacillus stearothermophilus glycoprotein from Methanocaldococcus jannaschii is highly (Sara and Sleytr 2000). Two copies 01 a hyperthermostahle dependent on temperature and pH used for sample prepara protease are attached to the S-layer glycoprotein of Staplty tion (Akca 2004). Similarly, the electrophoretic mobility of lothermus marinus (Engelhardt and Peters 1998). The high the S-Iayer protein from the hypenhermophilic archaean molecular mass S-Iayer fragment from Clostridium difficile, Nanoarchaeum equitans in SDS-PAGE depends on the tem obtained after specific post-translational proteolytic cleavage perature used for sample preparation .r Schuster et a1. 2004). of a precursor protein, expresses an N-acetylmuramoy1-L Aberrant migration behaviour has been also observed for the alanine amidase activity (Calabi et a!. 200 I). A metallopro lipid-modified S-layer glycoproteins from halobacteria ow tease from Caulobacter crescentus combines the cata ing to increased hydrophobicity (Konrad and Eichler 2002) lytic portion of a protease located at the N terminus with or to protein phosphorylation in the case of Aeramanas an S-layer-Iike protein at the C terminus (Urnelo-Njaka et al. hydrophile (Thomas and Trust 1995). A recent two-dimensional 2002). Finally, a unique ecological role for the cyanobacterial electrophoresis approach showed the prevalence of S-layer S-layer was shown for Synechococcus sp. strain GL24. Its and SLH proteins in the membrane fraction of a Bacillus hexagonal S-layer units function as discrete crystallization anthracis strain (Chitlaru et a1. 2004). Five new SLH pro nuclei for the biomineralization of gypsum and calcite . teins were found, and most notably, the S-Iayer protein EA 1 (Schultze-Lam et a!. 1992; Smarda et a1. 2002 I. was present in a high number of isoelectric and mass variants. This situation is indicative of substantial post-translational Gene sequences modifications. Accordingly, the cell envelope of Methane sarcina tnazei is composed of a mosaic of several S-Iayer To date, about 400 species from all important taxa of bac like proteins (Mayerhofer et al. 19(8). teria and archaea have been shown to have an S-layer (Messner and Sleytr 1992; Smit et al. 2002). Recently. a cor Functions responding gene (NEQ300J was also found in the nanosized hypertherrnophilic symbiont Nanoarchacum equitans (Wa Although S-Iayers are almost ubiquitously found in bacte ters et a!. 2003), ·a representative of a new archaeal phylum ria and archaea, more knowledge is desirable about their (Huber et a!. 2002). NEQ300 codes for a glycosylated pro physiological role. The strong resistance of these simple bio tein with 941 amino acids and a relative mass of logical membranes to extreme environmental conditions, 103 920 Da. It possesses a 22 amino acid leader peptide and such as high temperatures, low pH, and high ionic strength five putative N-glycosylation sites. The protein subunits oc (Engelhardt and Peters 1998; Claus et a1. 2002), suggests cur as dimers and are arranged on a lattice with sixfold sym that they contribute to the stabilization and protection of metry and a centre-to-centre distance of 15 nm (Schuster et cells. This is especially true for the S-layers of extremophilic al. 2004). A survey of complete or partial S-layer gene se Gram-negative archaea. which are directly anchored into the quences of bacteria and archaea is given in Table 1. cytoplasmic membrane and represent probably the most an cient prokaryotic cell wall. In all other taxa, the S-Iayer Three-dimensional crystallization seems to be a more or less a remnant cell wall component and not essential for cell shape or rigidity. Owing to the high The occurrence of a regular crystalline structure on the energy needed to produce and translocate large amounts of surfaces of a microorganism can usually be easily demon protein across the cytoplasmic membrane, other essential strated by transmission electron microscopy after negative functions of the S-layer must have evolved. staining (Baumeister et al. 1990: Harris 1997; Sleytr et a1. The pores in the S-layer lattice present a barrier for macro 2001). However, their detection is sometimes difficult molecules in the 30- to 40-kDa range (Breitwieser et al. because they are masked by superimposed amorphous cell 1992; Sara et al. 1992). Thus, exoenzymes may be retained materials, In our investigations, S-layer sheets were rendered within a periplasma-like space and cell-lytic enzymes may visible when additional proteins were removed from, e.g .. be excluded. sporulating cells of Bacillus sphacricus and Bacillus 1fie S-layers of the Grarn-negatlves'pecies Aeromonas fusiformis by gentle ultrasonication. Antibodies generated salmonicida, Campvlobacter fetus. Aerotnonas serpens, and against the purified S-Iaycr protein of Bacillus [usiformis E3 Caulobacter crescentus shield the bacteria from predation by also gave strong signals with the corresponding S-layer sheet Bdellovibrio bacteriovorans (Koval 1993), Owing to their preparation obtained from spores. In agreement with this ob protective role against humoral and cellular immune defense, servation, the S-layer protein EAl of Bacillus anthracis has the S-Iayers of Aeromonas salmanidica, Ccunpylobacter recently been demonstrated to be a persistent contaminant of fetus, Bacillus cereus, Bacillus anthracis, and Rickettsia sp. spore preparations (Williams and Turnbough .II'. 2004).

@ ~005 NRC Canada 734 Can. J. Microbial. \/01. 51, 2005

Tahle 1. S-Jayer-homologous protein genes.

Systemaric Size grouping (amino Molecular Gene Nucleotide Protein Species (class) acids) mass (Da ) designation accesssion No. accession No. Hatobacterium halobium Halobacteria 852 89814 csg 102767° P08 198b Halobacterium sp. NRC-] Halobacteria 836 8807] csg AE005139a Q9HM69b Haloarcula japonica TR-l Halobacteria 862 89814 csg D87290a Q9C4B4b Haloferax volcanii Halobacteria 827 85 189 csg M628J6" P25062b Iviethanothermobactel' Methauobacteria 1755 101061 MTH716 AEOO0851 c /\/\R8'i22F thermautotrophicus Delta H Methanocaldococcus jannaschii Methanobacteria 558 60547 slmjl " AB] 1636" Q58232h DSM 26fil Methanocaldococcus jannaschii Methanobacteria 440 50745 MJ0954 U67539a Q583Mb D5M 26fi] Methunothermococcus Methanobacteria 559 59225 slmt]d A.T308554" Q8X235b thermolithotrophicus D5M 2095 /vIethanococcus vannielii D5M Methanobacteria 5fi6 59064 slm1'1d A.T308553(l Q8X234b 1224 Methonococcus voltae D5M Methanobacteria 5fi5 59707 sla M59200a Q50833b 1537 Methanococcus maripaludis 52 Methanobacteria 553 56644 slpB NC00579F NP98799SC Methanococcus maripcludis 52 Methanobacteria 575 589948 sip NC00579I c l\TP987503 c Methanororris igneus D5M56fi6 Methanobacteria 519 55669 slmiI" A.T56499SU Q6KEQ4b M ethnnosarcinan marei 5-6 Mcthanobacteria 652 68878 slgB X77929a Q50244b Methanosarcina acetivorans Methanobacteria 557 62086 slg AE01079;c AAM04705 c C2A Methanothermus fervidus DSM Methanobacteria 593 65481 slgA X58297" P27373b 2088 M ethanothermus sociabilis DSM Methanobacteria 593 65503 slgA X58296" P27374b 3496 Nanoarchaeum equitans Nanoarchaeora 94] ] 03920 NEQ300 AEOl799' AAR39148c Euryarchaeta 37Fll Uncultured marine 564 61 747 AAF97179' AAF97179c group II Pyrococcus abyssi Orsay Thermococci 604 65985 A124828Y CAB49670' Pvrococcus horikoshii OT3 Thermococci 236 26128 PH1395 BA000001 c BAA30501' Tliermococcus kodakaraensis Therrnococci 540 57367 AP006878c BAD85778c KODI

c Thermococcus kodakaraensis Thermococci 573 61 8] 9 APOO6878' BAD84353 ' KODI Thermococcus kodakaraensis Thermococci 612 66631 AP006878e BAD85778c KOD] SulfolobliS acidocaldarius DSM Crenarchaeota 1424 151 040 sip] BNOO0824° CPOOOO77" 639 Thermoprotei SulfoJobales Sulfolobus solfataricus P2 Crenarchaeota 1231 131 868 slpI BNOOO829a CA.T31324a Thermoprotei Sulfolobales Staphvtothermus marinus FI Crenarchaeota 1524 166 287 U57967' AAC44118' Thermoprotei DesulfurococcaJes Th ermotoga maritima MDB8 Thermotogae 456 51807 NCOO0853' NP228650c Deiaacaccus iadiaduxans Sark Dei!1.Qcocci 1036 108028 hpi MI789S" P13 126 b Deinococcus radiodurans Deinococci J 167 J23 729 DR2508 AE002080a P56867b c Tliermus thermophilus HB8 Deinococci 917 96133 slpA X57333 P35830' Thennus thennophilus HB27 Deinococci 469 52219 NC005835 c YP005376' Gleobacter violaceus PCC 742] Cyanobacteria 697 721]J GIl4200 AP006582(l BAC92141 b Svnechococcus lividus PCC7942 Cyanobacteria 521 57139 somA DM077a P7754b Synechococcus sp. PCC 630 I Cyanobacteria 525 56213 somB U702564' AAC33402' Svnechococcus sp. PCC 6301 Cyanobacteria 532 57386 somA U702564' AAC33403c

Table 1 i continuedi.

Systematic Size grouping (amino Molecular Gene Nucleotide Protein Species (class) acids) mass (Dal designation accesssion No. accession No. Svnechocvstis sp. PCC 6803 Cyanobacteria 630 67600 s/r184J D90906" BAA17449b Svnechocvstis sp. PCC 6803 Cyanobacteria 321 34543 s/r2000 D90910a BAA17888h Caulobacter crescentus CB 15 o.-Proreobacteria 1073 103 613 rsaA AE005779c AAK22991 c Caulobacter crescentus 1S3001 rx-Prcteobacterin J026 98 132 rsaA AF193063a P35828h Caulobacter cresecntus 1S4000 c- Proteobacteria 359 35434 rsaA. AF193064" Q9RM.'\1 h Caulobacter vibriodes o.-Proteobacteria 658 68471 sl1pA AYOfi421I c AAL47190c Rickettsia japonica YH (X -Proteobacteria 1656 168098 rOmpS AB003681 a Q06653 h Rickettsia prowarekii Brein 1 (X- Proteohacteria 1643 169854 spaP M37647" Q53020 h Rickettsia rickeitsii j{ (X- Proteobacteria 1300 132802 1'120 X16353" Q53047 b Rickettsia tvphi Wilmington (X-Proteobacteria 1M5 169698 slpT L0466l a P96989b Desulfotalea psvchrophila LSv54 &- Proteobacteria 504 56155 NC006138c YP064946c Aeromonas hydraphila TF7 y-Proteobacteria 472 47646 ahs/; L37348 a Q44072 h Aeromonas salmonicida A450 y-Proteobacteria 502 52869 vapA. M64655 a P35823h Serratia niarcescens Sr41 y-Proteobacteria 1004 102406 s/aA AB00712SO Q54455 b Camplobacter fetus subsp. fetus s-Proteobacteria 942 96 634 sap-; .I05577a Q841Z1 h 23D Camplobacter fetus subsp. fetus r-Proteobacteria 922 95 J09 sapAJ LJ5800a Q841X3 h 23B Camplobacter [etus subsp. fetus r-Proteobacreria ll09 111 805 sapA2 S76860° Q53505 b 82-40LP3 Camplobacter fetus subsp. fetus e-Proteobacreria 811 88 153 sapAp8 AA0642l4c Q84177b 23D Camplobacter fetus subsp. fetus s-Proreobacteria 1167 117324 sapA3 AA06423l' Q84lXl b 23D Camplobacter fetus subsp. fetus E.- Proteobacteri a 1106 111 525 sapA5 AA06421 F Q841Z0 b 23D Camplobacter fetus subsp. fetus s-Proreobacreria 1257 129 306 sapA6 AA064213c Q841Y8b 230 Cantplobacter fetus subsp. fetus E-Proteobacteria 1292 130736 sapA7 AA06421SC Q841Y6b 23D Camplobacter fetus subsp. fetus r-Proteobacteria 939 95508 sapA A37284a Q841X5b 23D Caniplobacter fetus subsp. fetus fe Proteobacteria 1] 12 112504 sapS D::5133 Q Q46037" 84-91

Q Camplobaeter fetus subsp. fetus E-Proteobacteria 1112 112 504 sapli? AF048699 Q52781 b CIP 5396T Camplobacter rectus 314 E-Proteobacteria 1361 144386 Crs AF010143a Q30524 b Camplobacter rectus ATee e-Proteobacteria 1361 144904 ABOO1876a Q87083 b 33238 Camplobocter rectus ATee E-Proteobacteria Il23 118 954 esxA. AF035193 a Q9ZIB3 b 33238 Clostridium thermocellum NCIB Clostridia 1664 178 J94 eipA X67506 Q Q06852h 10682 Clostridium thermocellum NCIB Clostridia 688 74970 X67506 a Q06853 h 10682 Clostridium acetobutvlicum Clostridia 439 48 116 CAC3558 AEOO785Y AAK81482c DSM 792 Clostridium acetobutylicum Clostridia 1939 210 335 CAC3389 AE007836c AAK81319c DSM 792 CloSt!iiiltlm leU/ni E:5:5 Clostridia 354 37516 AE015937c AA035145' Clostridium tetani E88 Clostridia 642 72 100 AEOl5937c AA03510F Clostridium tetani E88 Clostridia 708 77200 AE036592c AA036592c Clostridium tetani E88 Clostridia 1334 145 565 AE015937c AA035096c Clostridium tetani E88 Clostridia 642 72 100 NC004557 c NP781164c Clostridium diffieile MRY04 Clostridia 335 35623 slpA. ABl81350c BAD2275JC 0409

Table 1 t continuedi.

Systematic Size grouping (amino Molecular Gene Nucleotide Protein Species (class) acids) mass (Dal designation accesssion No. accession No. Therrnoanaerobactcr kivui Clostridia 702 76511 M31069" AAA21930c Aneurinibacillus Bacilli 759 RI 431 .latA AY39557R c AAS44591 c thermoaerophilus L420-9] T Aneurinibacillus Bacilli 738 78306 sailt AY395579 c AAS4459Y thermoarrophilus DSM 10155 Bacillus anthracis Ames Bacilli 862 9] 362 eag X99724a P94217!J Bacillus anthracis Ames Bacilli 814 86620 sap Z36946a P4905]b Bacillus lichenifonnis NM ]05 Racilli 874 92735 olpA 038842a P49052b Bacillus pseudofirmus OF4 Bacilli 93] 96855 slpA AF242295Q Q9L655 b Bacillus sphaericus PI Bacilli 1252 129935 A45814a CAA02847b Bacillus sphaericus WHO 2362 Bacilli 1176 ] 25226 M2836F P38537b Bacillus sphaericus CCM 2177 Bacilli 1268 132047 sbpA AF211170a Q9RER7b j Bacillus sphaericus JG-A12 Bacilli 184 19687 A1292965" Q9EXR5b Bacilills sphaericus NCTC 9602 Bacilli 1228 129728 szt4 AJ866974c CA129282c Bacillus cereus DSM 31 Bacilli 577 64599 BC0991 AE01700F AAP0797SC Bacillus cereus DSM 31 Bacilli 530 58834 BC0902 AE017000c AAP07889c Bacillus cereus DSM 31 Bacilli 4]0 45571 BC3524 AE017009c AAPI0458c Bacillus cereus ZK Bacilli 578 64382 NC006274c YP082487c Bacillus cereus ATCC 10987 Bacilli 272 30467 NC003909c NP981245c Bacillus thuringiensies subsp. Bacilli 821 87123 slpA A1249446" Q9REDOb galleriae NRRL 4045 Bacillus thuringiensies subsp. Bacilli 393 45437 sip X62090a P35826b . israelensis 4Q2 Bacillus thuringiensies subsp. Bacilli 816 87293 etc A.1012290a Q9ZES5b finitimus CTC Bacillus thuringiensis serovar Bacilli 578 64750 NC005957c YP035240c konkudian 97-27 a e Bacillus fusiform is DSM 2898 Bacilli 576f 62 007j .1_rr A.1781693 . Q6EV58b ., Bacillus fusiform is B3 Bacilli 575f 61 874' ar A.1781692".e Q6EV59b e Brevibacillus brevis HDP31 Bacilli u is 123 397 Invp D90050a P38538b Brevibacillus brevis 47 Bacilli 1084 120768 mwp M19]15a P06546b Geobacillus stearothermophilus Bacilli 1099 115 395 sbsC AF055578° 068840b ATCC 12980 Geobacillus stearothermophilus Bacilli 903 96200 sbsD AF228338° Q9KIQ5 b ATCC 129801G+ Geobacillus stearothermophilus Bacilli 903 96640 sgsE AF328862c AAL46630 c NRS 2004/3a f a e b Geobacillus stearothermophilus Bacilli 439 4708:;! .Ill A.1781694 • . Q6EV57 .e DSM 2358 Geobacillus stearothermophilus Bacilli 1218 131 075 sbsA X71092° P35825b PV 72/p6 Geobacillus stearothermophilus Bacilli 920 97916 shsB X98095° Q45664b PV nlp1 Geohacillus kaustopliilus Bacilli 969 101668 NC006510' YPJ49039c HTA426 Lactobacillus acidophilus ATCC Bacilli 444 46570 slpA X8937SO P35829h 4356 Lactobacillus acidophilus ATeC Bacilli 456 47773 slpB X89376° Q48508b 4356 Lactobacillus brevis ATCC 8287 Bacilli 465 48159 slpA Z14250° Q05044b Lactobacillus brevis ATCC Bacilli 483 50924 slpB AY040846c AAK84948c 14869 ... ·slpC----- c Lactobacillus brevis ATCC Bacilli 461 48856 '-AY040847 AAK84949c 14869

Tahle 1 tconciudedv.

Systematic Size grouping lamino Molecular Gene Nucleotide Protein Species (class) acids! mass IDa) designation accesssion No. accession No. Lactobacillus brevis ATCC Bacilli 413 44572 slpD AY040848r AAKR4950' 14869 Lactobacillus crispatus lCM Bacilli 440 46771 cbsA AFOOI313" Q07120" 5810 Lactobacillus crispatus lCM Bacilli 452 47602 cbsB AF07936Y Q86015" 5810 Lactobacillus crispatus LMG Bacilli 458 48750 sipNA AF253043" Q9L5C3" 12003 Lactobacillus crispatus LMG Bacilli 439 467'J6 slpNB AF253044Q Q9L5C2" 12003 Lactobacillus fermentum BR 11 Bacilli 264 28627 bspA U97348° Q06530" Lactobacillus helveticus lClvI Bacilli 388 40899 AB061777c BABn067c 1007 Lactobacillus helveticus lCM Bacilli 391 41 199 AB061778c BAB72068c 1008 Lactobacillus helveticus ATCC Bacilli 439 46702 AJ388558" Q9S3AO" 12046 Lactobacillus helveticus ATCC Bacilli 439 46716 AJ388559Q Q9S399" 15009 Lactobacillus helveticus CNRZ Bacil1i 439 46688 A13885600 Q95398" 303 Lactobacillus helveticus CNRZ Bacilli 437 46485 AJ38R561 Q Q9XBI9" 35 Lactobacillus helveticus IMPC Bacilli 439 46704 AJ388562Q Q9S397" i60 Lactobacillus helveticus IMPC Bacilli 439 46602 A.I388563" Q9S396" M696 Lactobacillus helveticus IMPC Bacilli 438 46622 AJ38S564Q Q9S395" HLMI Lactobacillus helveticus CNRZ Bacil1i 439 46688 slpHl X91199a P38059" 892 Corynebacterium glutamicum Actinobacteria 510 55425 csp2 X69103a Q04985" ATCC 17965 Propionibacterium acnes Actinobacteria 463 48114 NCOO6085 c YP056872c KPA17l202 Pirellllla sp. 1 Planctomycetacia 524 55241 BX294138c CAD72961 c Pirellula sp. 1 Planctornycetacia 818 90680 burB BX294149c CAD7635SC Leptospira interrogans 56601 Spirochaetes 527 59704 slpM NC004342c NP71086c Leptospira interrogans LI-130 Spirochaetes 527 59693 NCOO582.3c YP00286SC Bacteroides thetaiotaomicon Bacteroidetes 393 44178 NC004663 c NP80936Y VPI-5482 Fusobacterium nucleatum ATCC Fusobacteria 643 71502 FN0694 AEOJ0580c AAL94890c 25586 Fusobacterium nucleatum subsp. Fusobacteria 643 71874 FNV1357 AABFOJOOO03SC EAA24374c vincentii ATCC 49256 Fusobacterium nucleatum subsp. Fusobacteria 640 71489 NZAABF0200003SC ZPOO 144025c vincentii ATCC 25586 Cytophaga sp. Jeang 1995 Sphingobacteria 1047 108718 AF06R060Q Q9RB95" Note: The systematic grouping was performed according to Garrity and Holt (200I). "ENtBr database. "Unil'rot/Swissf'rot database. 'GenBanklNCBI database. dAk9a et al. (2002). "Akca (2004). "Incomplete sequence.

Treatment with lysozyme has been described as a simple rl strategy for rapidly screening for the presence of S-layers on rl Gram-positive cells by transmission electron microscopy or scanning force microscopy (Wahl et a1. 20011. Electron ho lography of nonstaincd bacterial S-Iayer proteins has been applied to avoid possible structural artefacts caused by the heavy metal treatment (Simon et a!. 2004). Freeze-etching preparation of whole cells is another we1l-established method for the characterization of S-Iayer lattices (Sleytr and Glauert ] 976; Rachel et a!. 1986). Atomic force micros copy is a powerful tool for studying biological molecules in their native environment and has already been successfully applied to study the conformation and crystallization dynam ics of S-Iayers (Muller et a!. 2002; Gyorvary et al. 2003). The topography of S-Iayers can be readily modelled by three-dimensional reconstruction from electron micrographs tr-, r' (Baumeister et al. 1990; Beveridge et al. 1990; Lupas et al. 1994). Nevertheless, X-ray crystal structures have therefore been obtained only for small recombinant S-layer fragments .~ '0 from the hyperthermophilic species Staphvlothermus marinus v (Stetefeld et al. 2000), Methanosarcina marei (ling et 31. 00 r-. :;) 0'" on Q \~ r'l C", rl 2002), and Geobacillus stearathcrmophilus ATCC 12980 (Pav "C/O 31c, "1' 7 .,f -r -i kov et al. 2003). To obtain S-layer proteins for biochemical and biophysi cal studies, they are isolated after breaking whole cells by »<. ~ '" '"'~ vr, .,. t- sonification, removal of the cytoplasmic membrane with Tri :,) c. r-. \0 7 c- ~ C/O X ;::: OJ: '-C v-, rJ ton X-IOO, extraction, and dissociation into monomers by Q or, e 0\ 0\ 0\ treatment with high molar concentrations of chaotropic ;:;:: 2 or, «: ir-, or, or, agents (urea. guandinium chloride) or by cation substitution, 2 e.g., Na" or r.i" replacing Ca + (Sleytr et al. 20OJ). After re moval of the chaotropic agent by dialysis at neutral pH val g ues. the S-layer monomers reassemble into water-insoluble ;::

5 ~ materials. Repeated rounds of extractions and dialysis re OJ: v J 0\ oc 0\ .Q sult in highly purified S-layer proteins, which can represent N tr; or, \:) '2 ~ - v-, up to 15% of the whole cell protein. Further purifications (/J on v". 'r, 'r, can be achieved for example by preparative isoelectric fo

cusing, which has been recommended to improve protein ~,

=: ~ -.. -..-, -.. quality for crystallization experiments (Bott et al. 1982). .g ~ . Using these strategies and under low-gravity conditions, we a ec ~. -.. -:: -e-, "0 ~ ~ Z ~ ~ '" obtained protein crystals and the first X-ray data of complete ~ ::; C/O Ir, -c: -T <", v 0\ rr: Ir, .r, archaeal and bacterial S -layer proteins (Evrard et al. ]999; .2 ":J 0\ -s: Ir. v-, r, e", <", r. II, :) V ...., ....., ....., -< '::l methanococci e.. Cells of the order Mctlianococcales are covered by a sin 2 -5 ::: gleS-layer in a hexagonal arrangement that is directly ~ 0 P- 00 sr, v-, t- t exposed to the environment and cannot be stabilized by cel O c 00 ex: '-C r:-r! r"". lular components. As Methanococcales comprise species living at extremely different temperature conditions, their

S-layer proteins represent an ideal model for studying the ~

~ of-...r*ot~in ::: .- mulce oJar mechanisms stabilization at elevated ~ .:;: '" ~ ' ~ ~ temperatures. Molecular data of S-Iayer proteins from Meth ~ ;. .~ ~ ' ~ anococcales deduced from their gene sequences are com g ~ ~ .~ .s ~ 2 ~ -e; " piled in Table 2. t ~ -':: ~ ~ ":0 2 CJ CJ ~ " More predicted N-glycan sites have been found in the .2 ....I C/O '" ~ :) § '§ g r ~ I:C S-Jayer primary sequences of the hyperthermophilic Meth ':;.; -J ~ 'S -;::" -:: -':: 2: anocaldococcus jannaschi compared with its mesophilic rel g,. ..:::'.., " -s '";: ~ ~ ~ .", ..,. - -e-, ~ '" ~ ~ atives (Akca et a!. 2002; Claus et al. 2002). The same was o: -e "'"

Tahle 3. S-layer homologous proteins of the Bacillaceae.

Amino acid composition Molecular Leader peptide SLH Accession Species Nonpolar Polar Acidic Basic mass (Da) pI (amino acids) domains No. a

~ Bacillus cereus ATCC 14579 39.9 37.5 10.2 12.4 64599 8.34 29 .J AAPm978 Bacillus anthracis Ames Plasmid 42.5 31.2 12.9 13.5 45044 6.3 29 3 AAD32358 Genome 47.7 27.5 12.2 12.4 91 362 5.7 29 3 CAA68063 Bacillus lichcniformis NM lOS -17.8 25.5 13.4 132 92 735 5.71 29 .'~ JC4r)3 n Bacillus thuringiensies NRRL4045 47.4 24.1 14.7 l3.7 87 123 543 29 3 CAB63252 Bacillus pseudofirmus OF4 47.8 34.9 10 Ii 6.6 96855 4.42 31 3 AAF68431i Bacillus sphaericus DSM 396 48.3 32.3 9.6 9.9 85509 5.56 31 3 A.T292964 CCM 2177 48.2 34.8 7.4 9.6 132047 4.80 30 3 AAF22978 PI 48.6 32.9 8.3 10.5 129935 4.85 30 3 CAA02847 WHO 2362 44.2 29.2 12.3 ]4.3 125226 5.05 30 3 A33856 Bacillus [usiformis B3 47.1 28.2 11.3 13.4 61 874b 5.01 30 3 AJ781692b DSM 2898T 47.4 27.7 1l.4 13.2 620mb 5.09 30 3 AJ781693h Geobacillus stearothermophilus ATCC 12980 46.3 30.5 114 116 ] ]5 395 5.72 30 0 AAC12757 NRS 2004/3a 45.0 318 119 ] 1.3 96640 6.67 30 0 AAL46630 DSM 2358 44.9 30.5 13.9 10.7 47082b 9.18 30 0 AJ781694b PV 72/p6 45.2 31.4 113 12.0 131 076 5.40 30 0 140468 Brevibacillus brevis HDP31 41.0 27.7 17.2 140 123 397 4.85 53 ::' A35129 47 40.6 24.6 20.0 14.6 120768 4.65 24 ::' A28555 "EMBL database. "Incornplere gene sequence (Akca 20(4).

found for the S-layer protein of the hyperthermophilic Methexhibited an unusual electrophoretic behaviour in response anotorris igneus (Akca 2004), suggesting a role for glyco to cations, pH. and temperature. Whether similar confor sylation in the thermostabilization of these proteins. mational adaptations of S-layer proteins may occur under Although experimental data did not unequi vocally prove the extreme living conditions of archaea has to be investi S-layer glycosylation in hyperthermophilic methanococci, gated. indirect evidence for post-translational modification is sug The S-layer protein of Methanotorris igneus is 519 amino gested by the smaller size of the S-layer protein from Methacids in length. The presumptive 28 amino acid leader pep anocaldococcus 'jannaschii when heterologously expressed tide of the S-layer protein displays typical characteristics of in Escherichia coli compared with the native protein (Akca a signal sequence with a positively charged N-region, a hy 2004). On the other hand, glycans have been identified and drophobic core, a polar C-region, and an alanine residue at characterized in the hyperthermophilic methanogenic species the peptide cleavage site (Bendtsen et al. 2004). We found Methanothetmus fervidus and Methanothermus sociabilis that this leader peptide sequence is highly conserved (ap (Brockl et al. 1991; Karcher et al, 1993). proximately 68%) in all S-layer proteins of Methanococ Another signature of the hyperthermophilic methanococci cales, Overall sequence comparisons (e.g., by BLAST 2.0) Methanocaldococcus jannacliii and Methanotorris igneus is showed that the S-layer protein of Methanotorris igneus an increase in the number of charged residues, a decrease in shares 43% identity with Methanothermocacrus thermolitho alanine, and the occurrence of cysteine. Formation of disul trophicus, 39% with Methanocaldococcus jannascliii, 35% fide bridges, decrease in hydrophobicity, and increased gly with Methanococcus vannielii, and 34% with Methanococ e6~jlatiOI1 may CCJiIlrilmte to stability-of-hyperthermophilic CLlS voltae. archaeal S-layer proteins (Akca et al. 2002: Claus et aJ. The S-layer gene of Methaiiotarris igneus consists of 1987 2002). nucleotides (accession No. AJS64995) (Table 1). We identi The S-layer proteins of Methanocaldococcus jannaschii fied the putative promoter region including the transcription and Methanotorris igneus were purified by preparative iso factor B recognition element (ERE box, -GGTAA-) and TAT-'\. electric focusing (Akca 2004). The native and the recombi box i-TTTATATA-) at nucleotide positions ~3S and -28 up nant S-layer protein from Methanocaldococcus jannaschii stream from the transcription initiation site (-ATCG-). The

Table 4. Proposed regulatory gene sequences of the S-Iayer proteins lrom Bacillaceae.

Strain -35- box RihosOfl1e bindin~ ~ite itacillus anthracis Ames -1'1'G1'A1' -1'AC1'TT Bacillus licheniformis NM105 -1'1'G1'A1' -TACTTT Bocillus thuringiensis NRRL4049 -1'GTATG -TTCTAT Bacillus spluiericus DSM 2362 -1'TGAAT -TATAAT Bacillus sphaericus CCM 2177 -T1'GTA1'- -TATAAT Bacillus sphaericus PI Not found -TATAAT Bacillus [usiformis B3 -1'TGAAT -TATAAT Bacillu, [usiforinis DSM 28981' -TTGAAT -TATAAT Bacillus sphacricus DSM 396 -TTGCA1' -1'CAATT Geobacillus stearothermophilus DSM 2358 -1'AGCAC -1'AA1'A1\ Geobacillus stearotliermophilus A TCe 12980 -1'AGCAC -TAATAA

Shine-Dalgarno sequence (-AGGTGAT-) is localized down thuringiensis, (II! (al Bacillus sphaericus (CCM 2177, PI, stream at nucleotide +37. Translation starts (-ATG-) at nu WHO 2362). (h) Bacillus fusiforniis mSM 2SYST, B3), and cleotide +45 and stops with -VAA- at nucleotide +] 985. (e) Bacillus pseudofirmus, Bacillus sphaericus nSM 390, and Bacillus cereus ATCC 14579, (Ill) Geobacillus steam thermopliilus (ATCC 12980, NRS 200413a. DSM 2358), and S-Iaver of bacilli (IV) Brevibacillus brevis (HDP 31, 47). Much sequence information is available about the S-layer This division is also reflected by similarities within the zenes of the family Bacillaceae. A comparison of the corre corresponding regulatory gene sequences (Table 4). With re :ponding deduced 'primary protein sequences .wit~ respect to spect to the high diversity among Bacillus spp. and the rela biochemical and phylogenetical relatedness IS given 1I1 Ta tive few members included in this study, the taxonomic ble 3. All S-Iayer proteins exhibit signal peptides of 29-31 value of this grouping is limited. However. it is remarkable amino acids in size, except for two Brevibacillus brevis that group includes at least two phylogenetically related strains. This finding suggests a common phylogenetic devel r species (Bacillus anthracis and Bacillus thuringiensisi and opment of the leader sequences for translocation across the group III consists exclusively of the thermophilic members. cytoplasmic membrane. The proteins are generally weakly Group II comprises a genetically heterogenous group of meso acidic with the exception of the more baSIC proteins from philic round-spored bacteria. On the basis of 16S rRNA Bacillus cereus and Geobacillus stearothermophilus DSM analysis. Nakamura (2000) recently showed that Bacillus n58 (incomplete sequence). The molecular masses range sphaericus-Jike organisms segregate into seven phyJogeneti from approximately 45 to 132 kDa. In contrast WIth most cally different clusters. Data from Hastie and Brinton (1979) Geobacillus spp., one to three SLH domains have been found and Lewis et al. (1987) revealed that the proteins composing in all Bacillus spp. This is the only obvious difference in the the S-Iayer of Bacillus sphaericlls-like strains have a primary peptide structure between the thermophilic and the high moiecular mass (J 20-155 kfra), are slightly acidic mesophilic S-Iayer proteins of bacilli. When grown at 67 "C (pI 4.6-4.9), and have no or only Jow giycosylation (0.4- instead of 55°C. a variant of Geobacillus stearothertno 1.81 1110]%). The insectpathogenic strains possess a protein philus ATCC 12980 developed a glycosylated S-1ayer pro surface layer but lack the tetragonal arrays detected in the tein, whereas the wild-type protein is not glycosylated nonpathogenic strains by electron microscopy and negative (Ezelseer et a!. 2001). This observation may indicate a role staining (Lewis et a!. 1987). A possible relatedness between of glycans for thermostabilization of S-layer proteins. On th.e the S-layer protein and the parasporal crystals of insecticide other hand Novotny et a!. (2004) observed no change of eI strains is still controversial (Bowditch et al. 1989). ther the protein banding pattern or the glycan staining be haviour. Antibodies generated against the purified S-Iayer protein of Bacillus fusiformis strain B3 also gave strong signals with Overall sequence alignments generally exhibit no signifi that of Bacilln~ fusiform is DSM 2898. Only a weak cross re cant similarities among S-layer proteins of different species activity was obtained with the S-Iayer proteins isolated from and even strains of bacilli. However, this is not true for the Bacill;ls spliaericus DSM 396, Geobacitlus stearothermo N-terminal parts harbouring the domains for the membrane philus DSM 22, Geobacillus stearotlzermophilus DSM 458, transport, docking, and self-assembly processes: we found and Geobacillus stearothermopliilus DSM 2358 (unpub 95% homology (575 amino acids) between Bacillus fusi lished results). The differences in morphology and the [armis strains (B3, DSM 2898T), 94% (290 amino acids) be primary amino acid sequences of S-layers reflect the phylo .tvv eerr GC(jbctCilius st661rotlwrmfJphilus strains (ATCC 12980, genetical distance among the Bacillus-like bacteria. NRS 200413a. DSM 2358), 80% (J 92 amino acids) between Bacillus sphaericus strains CCM 2177, PI. WHO 2362), and 69% (201 amino acids) between Bacillus anthracis, Bacillus Conclusions licheniformis, and Bacillus tluiringiensis. On the basis of similarities in protein sequence and composition. the S-layer S-]ayer proteins are ancient biomolecnles that are almost proteins of Bacillaceae can be divided int~ four groups: (I) ubiquitously distributed in all prokaryotic taxa. Their intrin Bacillus anthracis, Bacillus licheniformis, and Bacillus sic ability to crystallize into two-dimensional lattices on the

cell surface may be one important step in the evolution of Boot. H..1 .. and Pouwe!s. P.H. 1996. Expression. secretion and anti ordered structures and early life. Archaeal S-Jayer proteins genic variation of bacterial S-!ayer proteins. Mol. Microbiol. 21: resist high temperatures and acidic, alkaline, and high-salt ]] 17-1123 environments. In bacteria that live under more moderate Batt, R.R., Navia, A.. and Smith, J.L J 982. Improving the quality conditions, S-layer proteins have been maintained but with of protein crystals through purification by isoelectric focusing. new physiological roles. In spite of the abundancy of S-layer J. BioI. Chern. 157: 9883-9886. proteins in many prokaryotic cells, these functions have Bowditch, R.D., Baumann, P, and Youston, A.A. ]989. Cloning and sequencing of the gene encoding a 125-kilodalton surface been explained in only a few cases. Apart from some con layer protein from Bacillus spliaericus 2362 and of a related served N-terminal structures, the majority of the high molec cryptic gene. J. Bacteriol. 17l: 4178-4] 88. ular mass S-layer proteins show no phylogenetic clusters Brcirwicscr, A., Gruber, K.. ami Sleytr. U.B. 1992. Evidence for an and heterogeneity is further enhanced by post-translational S-Iayer protein pool in the peptidoglycan of Bacillus stearo modifications. It is a challenge of future research to investi thermophilus. J. Bacteriol. 174: 8008-8015 Q.ate the mechanisms and meaning of this variability. Taken Brockl, G., Behr, M., Fabry. M., Hensel, R.. Kaudewitz. H.. together, the different S-layer proteins are exciting materials, Biendl. E.. and Konig, H. ]991. Analysis and nucleotide se not only for nanobiotechnological applications (Sleytr et al. quence of the genes encoding the surface-layer glycoproteins of 1997) but also as models to learn more about strategies for the hypertherrnophilic methanogens Methanothermus fervidus stabilization, self-organization, and functional evolution of and Methanothertnus sociabilis. Eur. 1. Biochern. 199: 147-152. proteins. Calabi, E., Ward, S., Wren, B., Paxton. T, Panico. M., Morris. H., Dell, A., Dougan, G., and Fairweather, N. 2001. Molecular char acterization of the surface layer proteins from Clostridium Acknowledgements difficile. Mol. Microbiol. 40: ] 187-1199. The authors want to thank the "Ministeriurn fur Bildung, Carl, M., and Dasch, G.A. 1989. The importance of the crystalline surface layer protein antigens of Rickettsiae in T-cell immunity. Wissenschaft, Forschung und Technologie (EMPT)" and the J. Autoimmun. 2(Suppl.): 8] -91. "Deutsches Zentmm fur Luff - und Raumfahrt (DLRj", Bonn, Chitlaru, T., Ariel, N., Zvi. A.. Lion. M., Velan, M.. Shafferman, A., Germany, for supporting these studies. and Elhanany, E. 2004. Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic References analysis: prevalence of proteins containing S-Iayer homology domains. Proteomics, 4: 677-691. Akca, E. 2004. Charakterisierung von S-Layer-Proteinen bei Pro Claus. H., Akca, E., Evrard, C.. Debaerdernaeker, T, Declercq, J.-P., karyoten. Ph.D. thesis, Johannnes Gurenberg-Universitat Mainz. and Konig. H. 2002. Primary structure of selected archaeal Mainz, Germany. mesophilic and extremely thermophilic outer surface layer pro Ak~a. E., Claus, H.. Schultz, N., Karbach, G.. Schlott, B., teins. Syst. Appl. Microbiol. 25: 3-12. Debaerdemaeker, T., Declercq, J.-P., and Konig, H. 2002. Genes Debaerdemaeker, T, Evrard. e., Declercq, J.P.. Claus, H.. Akca. E., and derived amino acid sequences of S-layer proteins from and Konig, H. 2002. The first crystaJJization of the outer surface mesophilic, thermophilic, and extremely thermophilic rnethano S-layer glycoprotein of the mesophilic bacterium Bacillus cocci. Extrernophiles, 6: 351-358. spliaericus and the hyperthermophilic 'archaeon Metliano Antikainen, J., Anton, L., Sillanpaa, J., and Korhonen, TK. 2002. thermus fervidus. Proceedings of the 2nd European Workshop Domains in the S-layer protein CbsA of Lactobacillus crispatus on Exo-/Astrobiology, Graz, Austria ESA SP-518, November involved in adherence to collagens, laminin and lipoteichoic ac 2002. ESA Publication Division, Noordwijk. pp. 441-442. ids and in self-assembly. Mol. Microbiol. 46: 381-394. Egelseer, E.M., Leitner, K.. Jarosch, M., Hotzy. C.. Zayni, S., Archibald, A.R., Hancock, r.c., and Harwood, c.n. 1993. CeJl Sleytr, U.B., and Sara, M. 1998. The S-layer proteins of the wall structure, synthesis and turnover. ill Bacillus subtilis and two Bacillus stearothermophilus wild-type strains are bound other Gram-positive bacteria. Edited by A.L. Sonenshein, .1.A. via their N-terminal region to a secondary cell wall polymer Hoch, and R. Losick. American Society for Microbiology, of identical chemica] composition. J. Bacteriol. 180: ]488 Washington, D.e. pp. 38]-410. 1495 Baumeister, W., Wildhaber, J., and Phipps, B.M. 1989. Principles Egelseer, E.M., Danhom, T, Pleschberger, M.. Hotzy, C; Sleytr, U.B., of organization in eubacterial and archaebacterial surface pro and Sara, M. 2001. Characterization of an S-layer glycoprotein teins. Can. J. 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