Bacterial S-Layers Uwe B

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Bacterial S-layers Uwe B. Sleytr and Terry J. Beveridge

ost Eubacteria and S-layers are produced by the self assembly and chemistry of prokaryotic Archaea possess a of proteinaceous subunits on the surfaces envelopes, S-layers have ap- Mwell-defined cell wall of prokaryotes, so that planar, parently coevolved with these outside the plasma (cytoplas- monomolecular-thick crystalline lattices diverse structures. In certain mic) membrane1 that presum- are formed. Some archaeal and eubacterial Archaea, the S-layer can be so ably evolved by selection in S-layer proteins are glycosylated. These closely associated with the response to specific environ- lattices typically have center-to-center plasma membrane that it is mental and ecological pres- spacings of less than 25 nm, which makes integrated into the bilayer. In sures. A common feature is the them attractive for biomimetic or Gram-positive bacteria, and in presence of a regularly ordered, nanotechnological applications. Archaea, the lattice assembles planar array of proteinaceous on the surface of the wall ma- subunits termed surface (S-) U.B. Sleytr is in the Center for Ultrastructure trix, which is composed mainly layers2. Because S-layers are Research and Ludwig Boltzmann Institute for of peptidoglycan or pseudo- Molecular Nanotechnology, University of ubiquitous and occur on both Agricultural Sciences, Vienna, Austria; murein, respectively. In Gram- Gram-positive and Gram-nega- T.J. Beveridge* is in the Canadian Bacterial Disease negative bacteria, the S-layer is tive bacteria (Fig. 1) and, when Network, College of Biological Science, University of attached to the lipopolysac- present, are one of the most Guelph, Guelph, Ontario, N1G 2W1, Canada. charide (LPS) component of the *tel: ϩ1 519 824 4120, abundant of cellular proteins, fax: ϩ1 519 837 1802, outer membrane. Some Gram- it is presumed they have a vital e-mail: [email protected] positive and Gram-negative function. However, a single bacteria can produce two super- function has been difficult to imposed S-layers; usually, each identify because S-layers fulfill many roles for the cell. is composed of a different subunit species. S-layers function as protective coats, as structures It is difficult to detect S-layered cells without using involved in cell adhesion and surface recognition, as electron microscopy and negative staining or freeze- molecular sieves, as molecular and ion traps, as a etching as preparative techniques (Fig. 2). High-resolu- scaffolding for enzymes and as virulence factors. In tion studies on the mass distribution of the lattices are Archaea such as Methanococcus, Sulfolobus and generally performed on negatively stained preparations Thermoproteus spp., which sometimes possess S- or unstained, thin, frozen foils. Two- and three-dimen- layers as exclusive cell-wall components outside the sional analysis, including computer-image enhance- plasma membrane (Fig. 1), the regular arrays even ment, has revealed structural information to ~1 nm determine cell shape and can direct cell division. (Refs 2–4,6). Recently, high-resolution images of the Because S-layers often have distinctive center-to- surface topography of S-layers were also obtained using center spacings, lattice formats and molecular weights, underwater atomic-force microscopy7, some images it was initially thought that they could be used as showing the dynamic opening and closing of channels taxonomic traits to distinguish bacteria at the species within subunits8. A common feature of S-layers is their level. Unfortunately, it was soon discovered that smooth outer surface and more corrugated inner strains within a single species could have distinctly surface. In S-layer lattices of Archaea, even pillar-like different lattices and that certain S-layers could even domains on the inner surface have been identified9. be altered at the level of the genome by environmen- S-layer subunits can be aligned in lattices with tal factors. However, because all S-layers share general oblique (p1, p2), square (p4) or hexagonal (p3, p6) features (all are made of relatively large proteins, self- symmetry (Fig. 2); hexagonal symmetry is predomi- assemble and are paracrystalline), certain sequence nant among Archaea. Center-to-center spacings of homologies should be conserved. Yet, comparative the morphological units can vary from 2.5 to 35.0 nm; studies indicate that S-layers are non-conserved struc- thus, the lattices can be quite porous, with pores tures and are of limited taxonomical value. Even occupying up to ~70% of their surface. The pores individual strains of selected species (e.g. Bacillus within an S-layer are of identical size (usually in the stearothermophilus) have been shown to be capable 2–8 nm range) and shape. Frequently, two or more of synthesizing different S-layer (glyco)proteins. distinct classes of pores can be present. Figure 3 shows how S-layers can be juxtapositioned with cell- Occurrence, location and ultrastructure envelope layers in Archaea and Eubacteria. S-layers have been observed on prokaryotes from nearly every phylogenetic group of Eubacteria and are Chemistry and assembly an almost universal feature of Archaea2–5. Despite the S-layers differ considerably in their susceptibility to fact that considerable variation exists in the structure disruption into monomeric subunits. They are

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PM (a) (b) PM (c) PG OM CW PM S S S

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Fig. 1. Electron micrographs of thin sections of (a) an archeon (Sulfolobus acidocaldarius), (b) a Gram-negative bacterium (Aeromonas salmonicida) and (c) a Gram-positive bacterium (Bacillus thuringiensis), all of which possess a crystalline cell- surface layer (S-layer). Abbreviations: CW, Gram-positive cell wall; OM, outer membrane; PG, peptidoglycan layer; PM, plasma membrane; S, S-layer. Scale bar ϭ 50 nm.

normally isolated from purified cell-wall fragments by lar biological structures. The isolated subunits assem- the addition of hydrogen-bond-breaking agents (urea ble spontaneously into regular arrays after removal of or guanidine hydrochloride), or detergents (at pH the disrupting agent used for their isolation. This <4.0) or by cation substitution (e.g. Naϩ or Liϩ replac- entropy-driven self-assembly forms low-energy struc- ing Ca2ϩ)10. Intersubunit bonds are stronger than tures and leads to lattices that are identical to those those binding the lattice to the underlying envelope observed on intact cells. Recrystallization can occur layer, a property that is seen as a basic requirement for with or without surface contact, and fragments have continuous recrystallization of the lattice during cell the ability to fuse into larger crystals. As in other self- growth. In some Archaea, the S-layers are very resist- assembly systems, the individual S-layer subunits ant to extraction, indicating the possible presence of contain all the information required for the growth of covalent intersubunit bonds4,5. a regular array. Chemical analysis and genetic studies of a variety of Studies on the in vivo morphogenesis of S-layers S-layers have shown that, with a few exceptions, they have focused on how the secreted subunits fold and are composed of a single, homogeneous protein or attach themselves to the underlying wall to form glycoprotein species with molecular weights ranging planar arrays during cell growth. Kinetic studies have from 40 to 170 kDa. These are often weakly acidic shown that with a cell-generation time of 20 min, proteins, typically containing 40–60% hydrophobic ~500 subunits per second must be synthesized, trans- amino acids, and possess few or no sulfur-containing located to the cell surface and incorporated into the amino acids. The pIs of these proteins range from 4 to 6, pre-existing S-layer lattice. The rate of synthesis of although the S-proteins from lactobacilli and Methano- S-layer proteins appears to be strictly controlled, as thermus fervidus have pIs ranging from 8 to 10. only small amounts are detectable in the growth A remarkable characteristic of many archaeal and medium of continuous cultures. Nevertheless, a few some eubacterial S-layers is their glycosylation11,12. species shed considerable amounts of S-layer15,16. The glycan chains and linkages are significantly Little is known about the specific interactions different from those of eukaryotes. The glycan of between S-layers and underlying wall components. In Halobacterium S-glycoprotein consists of short, pre- Caulobacter crescentus, attachment of S-layer sub- dominantly N-glycosidically linked sulfonated hetero- units requires Ca2ϩ and specific oligosaccharide-con- saccharides13. By contrast, glycans isolated from taining molecules17. Ca2ϩ is also required for adhesion Bacillaceae S-layers are assemblies of identical, re- in Campylobacter fetus subsp. fetus18. In this case, S- peating units with up to 150 monosaccharide residues, layer adhesion is via the amino-terminus of the pro- attached primarily by O-glycosidic linkages. Among tein, whereas in Aeromonas hydrophila strain TF7, them, novel linkage types, such as ␤-glucose→tyro- the carboxy-terminus is involved19. In other cases (e.g. sine, ␤-galactose→tyrosine or ␤-N-galactosamine→ Aquaspirillum serpens, strain MW5, Aquaspirillum threonine/serine, have been identified12. These struc- metamorphum, Aquaspirillum ordal and Bacillus tures are comparable with the LPS O-side chains of brevis strain 47) multiple S-layers can exist at the same Gram-negative bacteria14. time. These assemble sequentially, and subunits of S-layers provide a fascinating model for studying the outer lattice require the inner array as a template the dynamic processes of self-assembled, supramolecu- for assembly.

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(a)

(b) (c)

Trends in Microbiology

Fig. 2. Electron micrographs of freeze-etched preparations of intact cells of: (a) Thermoanaerobacter thermohydrosulfuricus (formerly Clostridium thermohydrosulfuricus) L111 showing a hexagonal (p6) surface lattice; (b) Desulfotomaculum nigrificans B200-71 with square (p4) lattice; and (c) Lactobacillus helveticus ATCC 12046 with an oblique (p2) array of subunits. ‘F’ are flagellae overlying the S-layer. Scale bars ϭ 100 nm.

In Lactobacillus buchneri20 and some B. stearo- Electron microscopy of freeze-etched preparations thermophilus species21, secondary wall polymers are and labeling experiments using fluorescent antibodies involved in S-layer interaction with the supporting or immunogold have revealed that different patterns layer. The S-protein of B. stearothermophilus strain of S-layer lattice extension exist for Gram-positive PV72/p2 has amino-terminal-binding domains for and Gram-negative bacteria. In Gram-positive bac- peptidoglycan and a secondary wall polymer. Mean- teria, lattice growth occurs primarily by insertion of while, other S-layer-homologous (SLH) domains multiple, helically arranged bands of S-layer on the have been identified, by sequence comparison, on cylindrical part of the cell. By contrast, in Gram- several S-layer proteins and cell-associated exo- negative bacteria, insertion of new subunits occurs at proteins22; these epitopes could also be involved in random. In all organisms, new S-layer lattices also ap- anchoring the proteins to the wall23. pear at regions of incipient cell division and the newly

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(a) (b)

S-layer S-layer Peptidoglycan and/or other Cytoplasmic polymers membrane Cytoplasmic membrane

(c) S-layer Membrane S-layer (glyco)- protein protein Outer membrane Peptidoglycan or Membrane lipids Peptidoglycan other polymers Cytoplasmic membrane Lipopoly- Lipo- Porin saccharide protein

Trends in Microbiology Trends

Fig. 3. Schematic illustration of the supramolecular architecture of the three major classes of prokaryotic cell envelopes containing crystalline bacterial cell surface layers (S-layers). (a) Cell-envelope structure of Gram-negative Archaea with S-layers as the only cell-wall component external to the plasma (cytoplasmic) membrane. (b) The cell envelope as observed in Gram-positive Archaea and Eubacteria. In Eubacteria, the rigid wall component is primarily composed of peptidoglycan. In Archaea, other wall polymers (e.g. pseudomurein or methanochondroitin) are found. (c) Cell-envelope profile of Gram-negative Eubacteria, composed of a thin peptidoglycan layer and an outer membrane. If present, the S-layer is closely associated with the lipopolysaccharide of the outer membrane.

formed cell pole. S-layers must be dynamic, energeti- to identical secondary wall polymers in both strains23. cally-closed surface crystals with the intrinsic ten- Interestingly, when a variant of PV72/p6 – strain dency to assume a continuously regular lattice during PV72/p2 – is induced during oxygen stress, a different cell growth. S-layer protein encoded by a different gene is produced, and the p6 lattice is converted to a p2 lattice Molecular biology, genetics and biosynthesis (Fig. 4). Remarkably, this S-layer variation is accom- Cloning and sequencing studies have brought panied by the synthesis of a new secondary wall considerable insight to the genetics and biosynthesis of polymer. In the S-layer protein of PV72/p2, a peptido- S-layers. Although the amino acid compositions of S- glycan-binding domain (between amino acids 1–138) layer proteins show no significant differences, this is and a new binding domain (between amino acids not true for the S-layer gene sequences; sequence hom- 240–321), both specific for the new wall polymer, are ologies of S-layer genes from Eubacteria and Archaea present23 . are rare7,24,25. Homologies can sometimes be seen be- Using sequence comparison, SLH domains have tween more closely related bacteria, for example, the been identified at the amino-terminal region of sev- S-layer genes from Lactobacillus acidophilus ATCC eral other S-layer proteins7 and at the carboxy-termi- 4356 and Lactobacillus helveticus have 80% homology nal end of other cell-associated exoproteins (e.g. in their nucleotide sequence, yet little homology was exoenzymes, binding proteins and pore-forming exo- observed with that from Lactobacillus brevis. toxins). Although only preliminary information on The homologies observed at the amino-termini of their structural interactions is available, these regions Gram-positive and Gram-negative S-layer proteins are predicted to be involved in anchoring the proteins are a result of the binding specificities for components to the cell surface. of the supporting wall layers (peptidoglycan, sec- Because S-layers are often the predominant pro- ondary wall polymers and LPS)7. For example, the teins in the cell, the promoters of S-layer genes must S-layer genes sbsA and sbsC from two closely related be very strong, particularly during growth at short B. stearothermophilus wild-type strains (PV72/p6 and generation times. For example, the promoter of the ATCC 12980) encode an identical amino-terminal S-layer gene from L. acidophilus is twice as effective27 residue composed of 250 amino acids. This homolo- as that of the gene encoding lactate dehydrogenase, gous region is responsible for anchoring the proteins which is one of the strongest promoters described in

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bacteria. In L. brevis, two promoters are involved in S-layer protein expression: one that is more closely located to the start S(p4) S(p2) codon and is mostly used during exponential and early-stationary- phase growth, and another promoter used at other times28. Ex- pression of B. brevis S-layers has been reported to require three promoters29. Trends in Microbiology Most S-layer proteins are secreted via the ‘signal sequence’ Fig. 4. Electron micrograph of the surface of a Bacillus stearothermophilus NRS 2004/3a cell, route, although for Aeromonas undergoing an oxygen-induced S-layer variation as the cells are grown in continuous culture; salmonicida, C. fetus and C. cres- the wild-type oblique (p2) lattice is eventually completely replaced by a square (p4) array. Scale bar ϭ 100 nm (modified from Ref. 26). centus, different secretion path- ways (such as an ABC or type I transporter system) have been described29. A few occur during continuous culture and, usually, the S- post-translational modifications are known to occur layer-deficient variant outgrows the S-layered parent. in S-layer proteins, including cleavage of amino- This implies that S-layers are only important in vivo. or carboxy-terminal fragments, phosphorylation of So far, a general, all-encompassing natural function amino acid residues and glycosylation30. for S-layers has not been found. It is possible that a An important trait of many S-layered bacteria is common S-layer motif has co-evolved in several dis- their ability to alter the S-layer protein, thereby lead- tinct prokaryotes to satisfy a variety of disparate ing to a specific cell-surface modification. Most de- needs. Alternatively, separate environmental pres- tailed studies have used C. fetus, which produces two sures on a single S-layered ancestor might have types of S-layer protein (designated A and B based on determined a diverse spectrum of different S-layer their binding to lipopolysaccharide serotypes A and functions. Most of the functional aspects that are B). Although C. fetus has only a single promoter, it is attributed to S-layers remain hypothetical. For exam- followed by eight to nine S-layer gene cassettes. This ple, the regularity of their lattice ensures an ordered pathogen is able to realign the promoter by a single system of constant-diameter channels over a bacterial DNA inversion, which, at frequencies that are inde- surface; this should exclude macromolecules of a pendent of the size of the DNA fragment, enables ex- larger size than the channel allowing the S-layer to act pression of different S-layer cassettes31. In this way, as a sieve. Such hypotheses have encouraged experi- the antigenic variation required to avoid a host im- mentation to show that S-layers can indeed function mune response is maintained by gene recombination. as molecular sieves and as molecule or ion traps in cell The observation that S-layers can be glycosylated adhesion or surface recognition, and in protection with several different glycans has led to studies on the from predacious attack by Bdellovibrio2,4,7. biosynthesis of prokaryotic glycoproteins (reviewed S-layers of several mesophilic and thermophilic in Ref. 11). In Halobacterium halobium, the synthesis Bacillaceae exhibit sharp exclusion limits between of the different N-linked glycans includes the transfer molecular weights of 15 000–40 000 Da (Ref. 2), of dolichol-linked saccharides to consensus sequences indicating a limiting pore diameter in the lattice of on the S-layer polypeptide for N-glycosylation. Simi- about 4 nm. Permeability studies have shown that the lar lipid-activated oligosaccharides with short-chain pores have excellent antifouling characteristics (i.e.

(C55–C60) dolichol species (rather than undecaprenol) do not become clogged), a feature considered essen- have also been observed in Haloferax volcanii and tial for the exchange of metabolites and nutrients. Methanothermus fervidus. Less detailed information These accurate molecular sieving properties might is available about eubacterial S-layer glycoprotein also prevent molecules such as lytic enzymes, comple- biosynthesis. In Paenibacillus alvei, dolichol (C55) ment, antibodies and biocides from entering the cell. carrier lipids have been identified, whereas in Thermo- Large molecules leaving the cell, such as hydrolases anaerobacter thermosaccharolyticum only nucleotide- and S-layer protomers, can also be trapped, thereby activated sugars have been characterized. In addition generating a functional equivalent to the periplasm, to nucleotide-activated monosaccharides, all pro- as seen in Gram-negative bacteria32. (In the Gram- karyotic systems also seem to require nucleotide-acti- negative bacteria the periplasm is trapped between vated oligosaccharides for S-layer glycan synthesis. the outer and plasma membranes.) Particular attention has been paid to S-layers as Function virulence factors on pathogenic bacteria. For the fish To keep a cell completely covered with an S-layer pathogen A. salmonicida, the presence of an S-layer requires a considerable biosynthetic effort. When provides resistance to the bactericidal activity of com- bacteria are no longer subject to natural environmen- plement in immune and non-immune sera33. As mental selection pressures, S-layers can be lost during re- tioned previously, the S-layer variation of C. fetus peated subculturing in the laboratory. This can also subsp. fetus helps this pathogen avoid phagocytosis

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and serum-mediated attack by the host’s immune sys- a series of glycoproteinaceous stalks (termed ‘tetra- tem31. Similarly, in Campylobacter rectus (a pathogen brachion’) which are branched and connected at the involved in human periodontal disease), S-layers top (Fig. 5). A four-stranded ␣-helical coiled coil inhibit adherence to fibroblast cells of the human makes up the stalk, to which two copies of the globu- gingiva and subsequent phagocytosis34. In Rickettsia lar protease are connected; the protease is an integral prowazekii and Rickettsia typhi, which are respon- part of the S-layer (Fig. 5) and has a molecular weight sible for epidemic and endemic typhus, S-layer pro- of 174 kDa, 24 kDa of which is made up of carbo- teins are strongly involved in humoral and cell- hydrate. Sequence homology suggests that the S. mediated immunity35. S-layers mediate cell adhesion marinus protease is a member of the subtilisin family for Clostridium difficile, an organism causing human of exoproteases. pseudomembranous colitis36, and Bacillus cereus37. S-layers have been shown to deter predation by Interestingly, virulent strains of Bacillus anthracis not Bdellovibrio bacteriovorus on several different cell only possess a capsule but also an S-layer, possibly types, including A. salmonicida, C. fetus and C. cres- consisting of two separate proteins, Sap and EA138. centus40. As the B. bacteriovorus predator must first Because S-layers have been reported on so many attach and break through the outer membrane of its pathogens of animals and humans, it is assumed that prey, it is thought that the S-layer is an additional they can be significant virulence factors. physical coating that inhibits attachment and entry. Recently, S-layers of Bacillaceae and Archaea were Yet, these same S-layers do not inhibit protozoa from found to function as adhesion sites for cell-associated ingesting Gram-negative bacteria41. S-layers can even exoenzymes9,39. The case of Staphylothermus mari- act as specific sites for bacteriophage adsorption, and nus is remarkable. This archaeon grows at high tem- phage resistance is mediated by variation of the perature (92°C) and has been isolated not only from relevant adsorption domain on the S-protein. a deep sea fumarole (or ‘black smoker’) from the East A remarkable function of the S-layer of Synecho- Pacific Rise but also from a hot submarine sediment coccus spp. is its role in inducing the precipitation of near Fossa volcano, Italy. It possesses a hyperthermo- gypsum and calcite from lake water42. To prevent stable protease on its S-layer9. This S-layer consists of their total encasement by a mineral layer, resulting in death, the cells shed patches of mineralized S-layer continuously, which eventually sink to the lake bot- tom, forming extensive marl sediments. This mineral- (a) (b) forming ability might be much more common among Arms prokaryotic organisms than is currently appreciated (reviewed in Ref. 43). 24 nm Analysis of cell morphology and the distribution of 32 nm lattice faults in the S-layers of Thermoproteus and Methanocorpusculum, genera that possess S-layers as STABLE 10 nm their sole cell-wall constituents, has provided strong evidence that S-layers can define cell shape and are involved in cell fission44,45. Cell division seems to be de- 70 nm termined by the ratio of the increase in protoplast vol- Stalk ume and S-layer surface area during cell growth. These observations, and in vitro self-assembly studies, have led to the speculation that S-layer-like membranes could have fulfilled barrier and support functions for self-reproducing systems at the origins of life. (c) Industrial applications S-layer As studies on the structure, chemistry, genetics, morphogenesis and function of S-layers have pro- gressed, their application potential has been recog- nized (for reviews, see Refs 2,46,47). To date, most Quasi- applications developed for using S-layers depend on periplasmic the in vitro self-assembly capabilities of isolated S-layer space subunits on the surfaces of solids (e.g. silicon wafers, polymers and metals), Langmuir-lipid films and liposomes. Moreover, because S-layers are periodic struc- Cell membrane Trends in Microbiology tures composed of a single (glyco)protein species and Fig. 5. The tetrabrachion–STABLE (Stalk-associated archaebacterial possess pores of identical size and morphology, they endoprotease) complex of Staphylothermus marinus and its organization exhibit identical physicochemical properties on each into an S-layer. (a) Electron micrograph of the complex, negatively molecular unit, down to the sub-nanometer scale. stained with uranyl acetate. (b) Schematic model of the complex, showing the dimensions of the components. (c) Schematic model of the cell Because the functional groups are aligned in well- surface. (Reprinted from Ref. 9.) defined positions and orientations, a broad spectrum of very precise chemical modifications can be applied.

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Currently, S-layers are used for the production of isoporous ultrafiltration membranes with well-defined Questions for future research molecular sieving and antifouling characteristics, and • Is there a common function for S-layers or are there many as matrices for the defined covalent attachment of separate, independent functions? functional molecules such as enzymes, antibodies, • Did S-layers evolve once on a common prokaryotic ancestor or protein A, biotin and avidin, as required for affinity several times on distinctly different ancestors? membranes, biosensors and solid-phase immuno- • Did S-layers first evolve on an archaeon? assays46,47. S-layers have also been used as supports • Why are some S-layer proteins glycosylated and not others? for Langmuir-lipid films or liposomes that mimic the • Why do some bacteria possess multiple S-layer genes or S-layer supramolecular surface architecture of archaeal or promoters on a single codon? • Can we take advantage of the natural periodicity of S-layers for viral envelopes. Self-assembly products are also suit- industrial purposes? able to potentiate an immune response when used as • Could S-layer-like membranes fulfill barrier and supporting func- carriers or adjuvants for antigens and haptens. It has tions as required for self-reproducing systems at the beginning been demonstrated that S-layers can be exploited in of life? nanostructure technologies and as matrices for controlled biomineralization. Finally, the incorporation of functional domains in S-layer proteins by genetic References approaches should lead to the development of new 1 Beveridge, T.J. and Graham, L.L. (1991) Microbiol. Rev. 55, types of recombinant vaccines, diagnostic agents and 684–705 biocompatible surfaces. The future of S-layer technol- 2 Sleytr, U.B. et al., eds (1996) Crystalline Bacterial Cell Surface Proteins, Academic Press ogy appears to be bright. 3 Sleytr, U.B. (1997) FEMS Microbiol. Rev. 20, 5–12 4 Beveridge, T.J. (1994) Curr. Opin. Struct. Biol. 4, 204–212 Conclusions 5 König, H. (1988) Can. J. Microbiol. 34, 395–406 There has been remarkable progress in S-layer re- 6 Hovmöller, S. (1993) in Advances in Bacterial Paracrystalline search during the past 25 years. In the early 1970s, Surface Layers (Beveridge, T.J. and Koval, S.F., eds), pp. 13–21, fewer than five laboratories were active in the field, Plenum Press captivated by the natural symmetrical beauty of these 7 Sleytr U.B. et al. (1999) Angew. Chem., Int. Ed. Engl. 38, arrays; now, there are well over 100 researchers, 4000–4020 and S-layers have been discovered on ~300 distinct 8 Müller, D.J. et al. (1996) J. Bacteriol. 178, 3025–3030 2,48 9 Mayr, J. et al. (1996) Curr. Biol. 6, 739–749 prokaryotes , with many more yet to be found. In 10 Koval, S.F. and Murray, R.G.E. (1984) Can. J. Biochem. Cell the 1970s, chemical extraction and purification tech- Biol. 62, 1181–1189 niques for S-proteins were laborious, crude and 11 Messner, P. (1996) in Crystalline Bacterial Cell Surface Proteins frequently inexact. Now, molecular-biological tech- (Sleytr, U.B. et al., eds), pp. 35–76, Academic Press niques are probing the most intimate details of selected 12 Schäffer, C. et al. Glycobiology (in press) S-layers and are finding, in fact, that few are closely 13 Sumper, M. and Wieland, F.T. (1995) in Glycoproteins related. S-layers that share almost identical lattice pa- (Montreuil, J. et al., eds), pp. 455–473, Elsevier Science rameters can have dissimilar nucleotide sequences. 14 Schäffer, C. et al. (1996) Microb. Drug Resist. 2, 17–23 Those that possess similar physicochemical charac- 15 Luckevich, M.D. and Beveridge, T.J. (1989) J. Bacteriol. 171, 6656–6667 teristics or (suspected) functional traits might not 16 Tsukagoshi, N. et al. (1984) J. Bacteriol. 58, 1054–1060 be related at all to one another. How rare in 17 Walker, S.G. et al. (1994) J. Bacteriol. 167, 6312–6323 biology! Clearly, since their early discovery in 1953 18 Dworkin, J. et al. (1995) J. Bacteriol. 177, 1734–1741 (Ref. 49), S-layers have intrigued researchers as they 19 Kostrzynska, M. et al. (1992) J. Bacteriol. 174, 40–47 attempted to understand S-layer protein synthesis, 20 Masuda, K. and Kawata, T. (1981) J. Gen. Microbiol. 124, transport, assembly, composition and function. But, 81–90 now, as we determine how genetically diverse these 21 Egelseer, E.M. et al. (1998) J. Bacteriol. 180, 1488–1495 symmetrical layers actually are, their fundamental 22 Lupas, A. et al. (1994) J. Bacteriol. 176, 1224–1233 importance increases: are they an example of parallel 23 Sára, M. et al. (1998) J. Bacteriol. 180, 6780–6783 24 Kuen, B. and Lubitz, W. (1996) in Crystalline Bacterial Cell evolution on a common structural theme, or an Surface Proteins (Sleytr, U.B. et al., eds), pp. 77–102, Academic example of extreme divergence from a single ancient Press structure? More importantly, how beneficial will they 25 Boot, H.J. and Pouwels, P.H. (1996) Mol. Microbiol. 21, be to the new applied fields of nanotechnology and 1117–1123 biomimetics? Those of us who initiated several of the 26 Sára, M. et al. (1993) J. Bacteriol. 176, 848–860 earliest S-layer studies are confident that Nature 27 Boot, H.J. et al. (1996) Mol. Microbiol. 21, 799–809 (through time, selection and evolution) has fabricated 28 Kahala, M. et al. (1997) J. Bacteriol. 179, 284–286 the best nanostructures of all! 29 Adachi, T. et al. (1989) J. Bacteriol. 171, 1010–1016 30 Bahl, H. et al. (1997) FEMS Microbiol. Rev. 20, 47–98 31 Dworkin, J. and Blaser, M.J. (1997) Mol. Microbiol. 26, Acknowledgements 433–440 Part of the work of U.B.S. was supported by the Austrian Science 32 Beveridge, T.J. (1995) ASM News 61, 125–130 Foundation Project Nr. S72, the Austrian Federal Ministry of Science 33 Trust, T.J. et al. (1993) Mol. Microbiol. 7, 593–600 and Transport and the Ludwig Boltzmann Society. The S-layer research 34 Borinski, R. and Holt, S.C. (1990) Infect. Immun. 58, of T.J.B. has been supported by the Natural Sciences and Engineering 2770–2776 Research Council of Canada (NSERC). 35 Carl, M. and Dasch, G.A. (1989) J. Autoimmun. 2, 81–91

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Filoviruses: closing the gap clearly delineates the differences system and the endothelium. on a killer between Marburg and Ebola Although some of the information viruses, as well as those between will undoubtedly be too detailed filoviruses and other negative- for the general scientific reader, Marburg and Ebola Viruses stranded RNA viruses. The fourth comprehension is aided by a series (Current Topics in Microbiology and fifth chapters, which contain of clear diagrams. The final chap- and Immunology series) previously unpublished data, pro- ter addresses the critical issue of edited by H-D. Klenk vide outstanding documentation filovirus virulence as an immune of the original Marburg virus out- response phenomenon. The au- Springer-Verlag, 1999. DM219.00/£84.00 hbk break and recent Ebola virus out- thor’s incisive review of the extant (vii ϩ 225 pages) breaks in Côte d’Ivoire and data on this topic suggests that the ISBN 3 540 64729 5 Liberia. The sporadic appearance unusually high mortality rates in of filovirus outbreaks and the dif- outbreaks of filovirus infection ficulties that have been encoun- might reflect an immunosuppres- ince their discovery in the tered in attempts to identify the sive effect by these pathogens. 1960s and 1970s, Marburg viruses’ natural hosts are well When multiple authors con- Sand Ebola filoviruses have covered. Clearly, without compre- tribute to a book, the chapters caused deadly outbreaks of hemor- hensive epidemiological infor- inevitably display a certain un- rhagic disease in Africa and mation of the type provided in this evenness in quality. Marburg and Europe. Because of their extreme book, future efforts to predict and Ebola Viruses is no exception. Par- pathogenicity, these often lethal, control filovirus outbreaks are ticularly bothersome is the dupli- negative-stranded RNA viruses doomed to fail. cation of coverage between chap- cannot be studied safely outside of The paucity of experience in ters emphasizing the pathogenesis a biosafety level 4 facility. None- treating filovirus infections has of filovirus infections. A minor theless, significant progress has hampered medical responses to complaint, but one reiterated by been made in understanding the localized outbreaks and has con- several members of my laboratory natural history, structure, repli- tributed to the high mortality staff, is that the book seems pecu- cative properties, pathogenesis figures associated with both liarly ‘old-fashioned’, both in and immunogenicity of filovirus Marburg and Ebola viruses. This terms of its cover design and its infections. deficit is addressed by chapters in textual appearance. Perhaps it is These advances are clearly set which specialists in the field con- time for Springer-Verlag to re- out in Marburg and Ebola Viruses. centrate on the clinical and patho- assess the visual impact of its Cur- As the Editor acknowledges in the logical aspects of Ebola virus out- rent Topics in Microbiology and Preface, the rationale for compil- breaks, using superb electron Immunology series, which has had ing this research was not based on micrographs, as well as histochemi- a long and illustrious history. any widespread immediate threat cal and immunohistochemical Overall, Marburg and Ebola posed by Marburg and Ebola micrographs, to illustrate their Viruses provided me with an en- viruses (the current number of narratives. Physicians will be es- joyable weekend of reading, and recorded human filovirus infec- pecially interested in sections I would have no reservations in tions is Ͻ1000), but rather on the describing the tools now available recommending the book to any ‘dramatic course of the disease, the for the diagnosis of filovirus specialist with an interest in the excess case-fatality rates, and the infections. filoviruses. lack of immunoprophylactic and The remaining chapters focus chemotherapeutic measures’. on knowledge gained from studies Yoshihiro Kawaoka Readers will appreciate the of experimental filovirus infec- Dept of Pathobiological Sciences, excellent overview of filovirus tions in animals, including a re- School of Veterinary Medicine, properties afforded by the first view of molecular findings that University of Wisconsin- chapter of the book. All of the suggest central pathogenic roles Madison, Madison, information is current, and it for the mononuclear phagocyte WI 53706, USA

TRENDS IN MICROBIOLOGY 260 VOL. 7 NO. 6 JUNE 1999