THE STRUCTURE OF CRYSTALLINE BACTERIAL SURFACE LAYERS Site Old PDFs/17-S-l… · STRUCTURE AND COMPOSITION OF BACTERIAL CELL ENVELOPES 1. Classification of Bacteria 2. Components

Prog. Biophys. molec. Biol., Vol. 51, pp. 131-163, 1988. 0079-6107/88 $0.00+ .50 Printed in Great Britain. All rights reserved. Copyright © 1988 Pergamon Press pie

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THE STRUCTURE OF CRYSTALLINE BACTERIAL SURFACE LAYERS

SVEN HOVMOLLER, AGNETA SJOGREN a n d DA NENG WANG

Department of Structural Chemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden

C O N T E N T S

INTRODUCTION 132

STRUCTURE AND COMPOSITION OF BACTERIAL CELL ENVELOPES 1. Classification of Bacteria 2. Components of Bacterial Cell Envelopes 3. Crystalline Surface Layers (S-layers) 4. Symmetries of Two-dimensional Crystals

METHODS FOR STRUCTURE DETERMINATION OF S-LAYERS 1. Electron Microscopy 2. Image Processing of Electron Micrographs 3. Three-dimensional Reconstruction

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136 136 137 140

THREE-DIMENSIONAL STRUCTURES OF S-LAYERS 1. Classification of S-layers According to Symmetry 2. Hexagonal S-layers

(a) Sulfolobus acidocaldarius (b) Thermoproteus tenax (c) Synechocystis sp. CLII (d) Eubacterium sp. AHN 990 (e) Clostridium thermohydrosulfuricum (f) Deinococcus radiodurans (g) Aquaspirillum serpens (h) Acetooenium kivui (i) Bacteroides buccae

3. Tetragonal S-layers (a) Bacillus sphae~icus (b) Sporosarcina ureae (c) Clostridium aceticum (d) Clostridium thermosaccharolyticum (e) Eubacterium sp. ES4C (f) Aeromonas hydrofila (g) Azotobacter vinelandi (h) Desulfurococcus mobilis Oblique S-layers (a) Methanospirillum hungatei

4.

REGULAR OUTER MEMBRANE PROTEINS 1. Porins 2. Other Regular Outer Membrane Proteins

OTHER CRYSTALLINE SHEETS OF COAT PROTEINS 1. Bacterial Spore Coats 2. Algal Cell Walls 3. Viral Coats 4. Gap Junctions

AMINO ACID SEQUENCES OF S-LAYER PROTEINS I. Homologies in Closely Related Strains 2. Sequences of Distantly Related Bacteria 3. Signal Sequences 4. Prediction of 3D Structures from Amino Acid Sequences

BIOLOGICAL FUNCTIONS OF S-LAYERS 1. Protection against Hostile Environment 2. Specific Adhesion

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132 S, H{)VM(}LLER PI ill.

3. Interaction with Bacteriopha#es 4. Shape Determining Coat

IX. BIOTECHNOL{)GICAL APPLICATI()NS 1. Ultrafihration Membranes 2. Genetic En,qineerimj

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X. CON('LUI)ING REMARKS l {~] ACK NOWLEI)GMEN'I'S [ (~ I REFERENCES 16 ]

I. I N T R O D U C T I O N

Many different bacteria have been found to have a regularly ordered protein layer as the outermost component of the cell envelope. This layer is called the surface layer or S-layer. The first report of an ordered array on the outside of a bacterial cell wall was in 1953, in a study of a Spirillum species by electron microscopy (Houwink, 1953). Since then several hundred different species of bacteria have been found to possess S-layers. A list of over 200 examples can be found in a review by Sleytr and Messner (1983). S-layers occur both in Eubacteria (Gram-positive and Gram-negative) and A rchaebacteria. As many as 10 or 20% of all bacterial species may possess an S-layer.

FIG, I. Freeze-etchcd preparat ion of Eubacterium sp. ES4C. The tetragonal :S-layer covers the v~holc surface of the elongated bacterium. F rom Sj6gren et al., 1988,

S-layers cover the entire bacteria, even during cell duplication (Fig. l I. They are composed of protein, usually a single polypeptide. The proteins may be glycosylated but are not embedded in lipids. They are arranged in a regular monolayer, making up a two-dimensional (2D) crystal. S-layers can be described as open networks, with pores often large enough to allow even small proteins to pass through (Messner et al., 1984). In order to cover the entire surface of a bacterium some hundred thousand S-layer monomers are needed, and in most bacteria with S-layers this will be the most abundant of all proteins in the whole organism.

Crystalline bacterial surface layers 133

Structures of S-layers from different bacteria have been studied by electron microscopy and computerized image processing. The S-layers studied have shown a wide variety of packings, with spacings from 5 to 33 nm and thicknesses from 5 nm and up. The most common symmetry is hexagonal, followed by tetragonal, but also trigonal and oblique arrangements have been observed. A review by Baumeister and Engelhardt (1987) is recommended.

The function of S-layers has not been studied as extensively as the structure, but the fact that this is the most abundant protein of the bacterium indicates that it must be of vital importance. Being the outermost part of the bacterium it will determine the surface properties and hence many of the processes involved in interaction with the environment.

In this review the structures of S-layers are described, based on data obtained mainly from electron microscopy and computerized image processing, but also from chemical characterization and DNA-sequencing. The biological functions and some biotechnological applications of S-layers are discussed.

II. STRUCTURE AND COMPOSITION OF BACTERIAL CELL ENVELOPES

1. Classification of Bacteria

The classification and taxonomy of bacteria is a science in its own right. Bacteria are first grouped into Archaebacteria and Eubacteria. Eubacteria are further divided into Gram- positives and Gram-negatives. These classifications are based on the cell envelope architectures.

For all three groups of bacteria a number of species with an additional layer of regularly arranged proteins, the S-layer, has been found.

2. Components of Bacterial Cell Envelopes

The bacterial cell envelope defines and encloses the single-cell organism. As such it must serve multiple purposes--it must be rigid and tight enough to withstand chemical and mechanical stress, at the same time keeping a relatively constant chemical composition inside the cell and allowing nutrients to pass selectively into and waste products out of the cell. The surface of the cell envelope will be important for cell adhesion, and also is the site of recognition for attacks by bacteriophages or by antibodies. Schematic drawings of the three main types of bacterial cell envelopes are given in Fig. 2.

Archoebocteria

(/~(~S-layer protein

Gram-positive Gram-negative

~ membrane pro1:e in D~ ,o~,n pep t i dog i ycan

lipopolysaccharlde ~ : ~ (or o t h e r po lymers )

FIG. 2. Schematic drawing of bacterial cell envelopes. In all bacteria a lipid bilayer with many different proteins enclose the cytoplasm. In Gram-positive and Gram-negative bacteria there is a peptidoglycan layer (the cell wall) outside the cytoplasmic membrane, and in Gram-negatives a second lipid bilayer, mainly containing porin, is found outside the peptidoglycan layer. In all three types of bacteria a crystalline array of proteins, an S-layer, may be found as the outermost component

of the cell envelope.

134 S. HOVM()LLER et Ol.

The main barrier against uncontrolled diffusion of solutes into and out of the cell is the lipid bilayer. Into this a number of amphiphilic membrane proteins are inserted, each with a specific function, such as transporting molecules or ions across this barrier. In the Gram- negative bacteria there are two such lipid bilayers, containing different proteins: in general the outer membrane, with porin as the main protcin, does not contain as many different proteins as does the cytoplasmic membrane. Furthermore lipopolysaccharides are found in the outer membrane. Peptidoglycans or other polymers contribute to the stability and resistance of the cell wall in Eubacteria.

Outside these components of the cell envelope there may be an additional layer of protein, the S-layer. Unlike hydrophobic proteins embedded in lipid bilaycrs, S-layer proteins arc generally hydrophilic with a net negative charge. They can often self aggregate into regular arrays after purification in L:itro, but in some cases the two-dimensional crystals will only form when the protein is supported by a membrane. In such cases the S-layer proteins may bc anchored in the underlying membrane by a short hydrophobic stretch of the protei.n.

The S-layers are usually composed of a single polypeptide of molecular weight ranging from some 40 to 300 kilodaltons tkl)a) (Sleytr and Messner. 1983). The proteins may bc glycosylated (Messner el al., 19841.

3. Crystalline Sm)/ace Layers ¢ S-layers )

The definition of S-layers has been somewhat unclear, but at the Second International Workshop on S-layers, held in Vienna 1987, the following definition was agreed upon: S-layers are surface envelope components on prokaryotic cells consisting of two-dimensional crystalline arrays of proteinaceous subunits (Sleytr et al.. 1988).

Apart from S-layers these structures have also been called RS-layers (Regular Surface layers), and there are also special names referring to the S-layer in specific species, such as the A-layer (Additional) of Aeromonas salmonicida, the HPI-layer (Hexagonally Packed Intermediate) of Deinocoecus radiodurans, the T-layer {Tetragonal) in Bacillus sphaericus. and the M W P and O W P (Middle and Outer Wall Proteins) of Bacillus brevis. Throughout this article the name S-layers will be used.

In general, S-layers form the outermost surface of a bacterium. However, in some cases there may be an additional layer of mucus outside the S-layer. In some bacteria there are quite similar-looking crystalline arrays in the outer membrane, such as porins and the crystals found in Chlamydia trachomatis (Chang et al., 1982) or even the crystalline array of bacteriorhodopsin found in Halohacterium halobium (Henderson and Unwin. 19751. The general agreement is not to include these regular outer membrane proteins (rOM Pt within the concept of S-layers. One differentiation would then be thai S-layers are hydrophilic proteins that self-assemble whereas the rOMPs arc hydrophobic membrane proteins that will aggregate only within a lipid bilayer. This borderline is unfortunately not as sharp as il may seem, since some S-layers have a minor hydrophobic part ~.hich serves to anchor the protein in a lipid bilayer. Finally there are the bacteria carrying two different layers of crystalline protein on their surface, and it may be discussed whether one or both of these is a proper S-layer.

Furthermore there are 2D crystalline arrays of proteins found m other organisms than bacteria. There are the coat proteins of viruses, surface proteins on algae and excn the gap junctions between eukaryotic cells. Perhaps one day' some of these ~ill be found to be closely related to the S-layer proteins, although at present they are not included in our delinition. For the sake of comparison a short account of such structures is given in Section VI of this review.

4. Symmetries qf Two-dimensional Crystal.~

S-layers may be classified on the basis of their symmetries. The crystalline nature of S-layers is an advantage for the analysis of their structure, since the powerful tools of crystallography can be applied.

There are 230 different ways of packing three-dimensional objects into three-dimensional (3D) crystals. These are the 230 space groups that are listed in the lntermitiomd Tahh's/br


Crystallography (1983). For two-dimensional objects packed into a plane there are only 17 possible crystal symmetries, the 17 plane groups. An interesting intermediate case is when 3D objects, such as proteins, form 2D crystals, such as the S-layers. Such crystals can be thought of either as 3D crystals only one unit cell thick, or 2D plane groups with a finite thickness. These intermediate crystals, 2½-dimensional if you like, are called two-sided plane groups. There are 80 of them, and they have been described by Holser (1958).

Most crystals contain the two different mirror images of the molecule (the two enantiomers) in equal proportion. Proteins are chiral (have a handedness) since they are composed of only L-amino acids. Out of the 80 two-sided plane groups only 17 do not have mirror symmetries, and only these are possible for proteins. Interestingly enough these 17 two-sided plane groups have a one-to-one relation to the 17 plane groups that will arise when these crystals are looked at in projection. S-layers are mainly studied by transmission electron microscopy and this technique gives a projection of the structure. Thus the S-layer crystals are arranged as two-sided plane groups, but the electron micrographs will show just the projected plane group symmetry. For the above reasons the crystal symmetries of S-layers are sometimes described as the corresponding 2D plane group seen in the images, sometimes as the 3D space group, and sometimes as the two-sided plane group. The latter of these is considered the most appropriate and is the predominant form in the latest publications.

All S-layers that have been studied in 3D have shown a difference between the two sides of the S-layer, making the crystals polar. This asymmetry makes sense biologically, since the protein molecules face different environments on the inside and outside, with one side of the S-layer facing the cell and the other facing the surrounding medium. If the S-layers fulfil a function involving directional movement, resembling pumping for example, they must be polar. This reduces the expected symmetries to be found for S-layers to only five, namely pl, p2, p3, p4 and p6. Indeed all S-layers that have been well characterized with respect to crystal symmetry belong to one of these five two-sided plane groups. The five expected symmetries are illustrated in Fig. 3, and the corresponding equivalent positions and symmetry relations between diffraction amplitudes and phases (see also Section III.2) are listed in Table 1. A complete description of all 17 two-sided plane groups may be found in Hovm611er (1986).

pl p2 p3

p4 p6

Fie. 3. The five two-sided plane groups expected to be found for S-layers. From Hovm611er, 1986.

136 S. HOVM~'iLLER et al.

III. M E T H O D S FOR S T R U C T U R E D E T E R M I N A T I O N OF S-LAYERS

1. E l e c t r o n M i c r o s c o p y

The two main specimen preparation techniques for electron microscopy used in studying S-layers are metal shadowing and negative staining. If the S-layers can be isolated without disintegrating, negative staining can be used. This provides a relatively high resolution (1.5--2.5 nm) of the projected protein structure (Fig. 4). Metal shadowing gives an image of only one surface, and usually with a poorer resolution (2 5 nm). However, in many cases where the S-layer is fragile, metal shadowing of intact bacteria is the only way to demonstrate the presence of an S-layer. In some cases the S-layer is covered by carbohydrate which must be removed, for example by lysozyme, in order to render it visible by any method.

TABLE 1. CHARACTERISTICS OF THE FIVE TWO-SIDED PLANE GROUPS THAT MAY BI FOUND FOR S-LAYERS

Symbol

Reflecticms Relations Symmetry of with phase between Equivalent diffraction restrictions

a and b axes positions pattern (being 0 ' or 180 )

pl (x, y,-) !F¢hkl!]- I FI/;~)I p2 .... (x, >,,z), (:~. >,,z) As for pl and ]flhkll[=[r~hld)[ thk¢))

p3 a = b , 7= 120 Ix, y, zl, (y, x - - v , z), As for pl and 6-fold symmetry I v - - x , 2, z) [F(hkl)[ = [F(k, h-+ k-, l ) ! - ]Fl l i + ~, h. I) i

p4 a = b , y=90' {x, y, z), (y, x, z), As for p2 and IF(hkl)[ = IFlkhlii lhk0) {x, .~, zt, O', x, zl

p6 a = b , 7=120 ( x , y , z ) , ( f , , x - y , z ) , As for p3 and ]r(hkl)l-=]FihkT)[ (hkt)) l y - x , 2, z), (2, y, z). (y, y - x , z), ~x )', x, zl

After Hovm611er (19861. Nomenclature and symbols are explained in International Tables lhr Crystal loqraphyl1983i .

Metal shadowing can be done on air-dried bacteria, but more commonly frozen bacterial suspensions are freeze-fractured and metal-coated after sublimation in vacuum (freeze- etching). These techniques have been described in detail by Baumeister et al. (1986a). The resulting images give a three-dimensional impression, since they depend on the surface relief of the specimen (Fig. 1). Albeit beautiful the structural information on a molecular level is usually limited, due to low resolution. More importantly, freeze-etched and metal-shadowed specimens provide a means to study biological objects in near in r i r o conditions, because the sample is deep-frozen in Freon in a fraction of a second. If the resolution is high enough, metal shadowing can also be used to distinguish between the inside and the outside of an S-layer.

The proteins of S-layers are not visible as such by electron microscopy because of the lo~v contrast provided by the light elements C, H, O and N that make up the polypeptide. An unprotected protein is destroyed by radiation damage within the time it would take to focus the electron microscope. Staining the sample with heavy metal salts solves both the problem of low contrast and that of radiation sensitivity, but at the expense of resolution. The heavv metal salts (most commonly uranyl acetate, phosphotungstic acid or ammonium molybdate I embed the proteins. What we see in the electron microscope is the difference in electron scattering from the protein and the heavy metal stain. The result is an outline of the protein, but no internal details such as alpha helices can be seen in stained specimens. During exposure the protein will be destroyed and the heavy metal salt will decompose, for examplc by losing crystal water and CO 2 from carboxyl groups in acetates. Finally only a metal oxide is left, but this is very stable in the electron beam, and it retains the outer shape of the protein molecules as a mould. The image is in fact just an image of the heavy metal stain distribution and the protein structure is deduced as having filled up the empty gaps in the stain. From this the method has its name negative staining.


i~i!ii ̧

F1G. 4. Negatively stained S-layers. (a) Bacteroides buccae, a Gram-negative bacterium. The S-layer can be seen to be associated with the outer membrane. From SjBgren et al., 1985. (b) Eubacterium sp. AHN 990, a Gram-positive bacterium, where the S-layer is detached from the rest of the cell envelope.

The symmetry is hexagonal, p6 in both cases.

The mass-loss of the protein and the stain upon electron irradiation causes a shrinkage of the specimen. This phenomenon evaded detection until very recently, because the collapse of the specimen is almost entirely in the direction parallel to the electron beam. The result is a thinner specimen, but the projected density remains largely unaffected. It was only careful work on three-dimensional crystals that revealed this shrinkage of negatively stained specimens (J6sior, 1982; Berriman et al., 1984). The obvious consequences of this shrinkage for 3D structure determination will be discussed in a following section (III.3).

2. Image Processing of Electron Micrographs

The regular lattice of a stained S-layer can be seen in the electron micrograph by the naked eye (Figs l, 4 and 5). If the S-layer is examined in greater detail in order to determine the

138 S. H(I',MOLLtl ~, ('1 al.

structure within each unit cell, it becomes clear that the noise level of the image is so high thai every unit cell looks different. In order to get an unbiased description of details within the unit cell it is necessary to obtain an average of images of many unit cells. When many unit cells are averaged the signal (structure) is enhanced relative to the noise. Such averaging was first done by optical filtering (Klug and DeRosier, 19661 and later by computerized image processing, as reviewed by Amos e t a / . (1982). Well ordered areas of S-layer are selected m an optical diffractometer tfor a simple setup see for example Hovm611er, 1986) and digitized using a microdensitometer.

Fic;, 5. Perpendicular section through the double S-layer of Lampropedia hyalina. ] 'he crystalline order is clearly seen. Piclure taken by J. W. Austin, Ontario.

The digitized area tFig. 6t of some one hundred unit cells is Fourier transformed and the resulting diffraction pattern (Fig. 7) is displayed oil a computer graphics screen, where the lattice is indexed. The sharp diffraction peaks represent the periodic features of the image, namely the protein structure, while the diffuse background represenls aperiodic noise. The noise can be filtered out by retaining only the information on the diffraction peaks. The diffraction pattern seen on a computer graphics screen represents only half the information present in the Fourier transform, namely the amplitude part. The other half of the information, the phases, is also calculated in the Fourier transform of the electron micrograph but not displayed. Unlike in X-ray crystallography where the phases cannot be determined directly because the diffracted beams cannot be focused, in the electron microscope the diffracted beams are focused into an image, and therefore the phases are not lost. The Fourier transh+rm of an imagc is a 2D function of complex numbers (A + iB) . The

amplitude (\. A - + B ~ ~ is what we see in the diffraction pattern. ] 'he phases [arctan(B/A ( + 180 degrees if A < 0 I] can also bc read out from the computer file. A set of amplitudes and phases {one such pair for every diffraction peak) is obtained, and is used as input to a standard crystallographic computer program to calculate a density map by the inverse Fourier transform (Fig. 8 I.


FJ~. 6. Digitiled area of the S-layer of Eubacterium sp. AHN 990, shown in Fig. 4(b). An area of 256 × 256 picture elements (pixels) each 40 x 40 um was scanned.

FIG. 7. Computer calculated Fourier transform of the digitized area shown in Fig. 6. The information from the crystalline S-layer protein is found at the sharp peaks of the hexagonal lattice, while the aperiodic noise is spread over the whole transform. The crystal axes h and k are marked. The

outermost diffraction peaks correspond to 18/k resolution.

Compu te r i zed image processing closely resembles opt ical filtering, but it can be taken further. The ampl i tudes and phases from every diffract ion peak are ob ta ined in numer ica l form, and these numbers can be further ana lysed and man ipu la t ed . Crys ta l symmet ry imposes re la t ions between ampl i tudes and phases for symmet ry - re la ted reflections. These rela t ions can be seen in Table 1. Even a filtered image still con ta ins some noise, since noise falling exact ly on a lat t ice poin t canno t be filtered out. The effect of this is to make ampl i tudes of symmet ry- re la ted reflections differ, and phases to move away from their true values. The ul t imate e l imina t ion of noise is therefore done by sett ing symmet ry - re la ted reflections to the average value of their ampl i tudes . Phases that for symmet ry - reasons have to be ei ther 0 or 180 degrees (see Table 1 ) are typical ly some 10 to 30 degrees away from these values. Phase

140 S. HOVM6LLER et al.

~ . , oO - ~ ~ , oO - p ~ - oO - / v ~ oO

-m T- oo T- oO -;o T-oo- v

~ - oO - ~ , - . ~ oO - ~ ~ oO - / v . _ ~ . , oO

FIG. 8. Projected density map of the S-layer of Eubacterium sp. AHN 990, obtained by inverse Fourier transformation after filtering out the noise in Fig. 7. This map represents the average of the 100 unit cells scanned. Hexameric star-like protein molecules are contoured. Unit cell dimensions

a = b = 15.7 nm. (a) Before and (b) after imposing exact p6 symmetry.

values around 0 degrees are then set to be exactly 0, and those around 180 are set to 180 degrees. The justification of this procedure can, and should, be tested by comparing amplitudes and phases from several images. Only reflections with consistent phases should be included in the calculation of the density map.

The improvement of the density map in Fig. 8(a) obtained by imposing the exact crystallographic symmetry may be appreciated from Fig. 8(b). Already in the un- symmetrized map (Fig. 8(a)) it is clear that the S-layer is hexagonal, but the final symmetrized density map (Fig. 8(b)) shows even clearer the protein net.

3. Three-dimensional Recons truc t ion

All biological objects are three-dimensional, and so are best represented in 3D. It is possible to reconstruct a 3D object from a set of projections taken from different angles of the specimen. In some favourable cases, such as helical structures, a single image provides at the same time all the views around the helical axis. As was shown by DeRosier and Klug (1968) a 3D reconstruction can then be obtained from a single image.

For a 2D crystal a different approach must be taken. The 3D structure can be reconstructed from a series of images taken at different angles of tilt in the electron microscope, as demonstrated for baeteriorhodopsin of Halobac ter ium halobium by


Henderson and Unwin (1975). A comprehensive review of the technique can be found in Amos et al. (1982). In summary the principal idea is to take one or more tilt-series of images from the crystals, with pictures taken about every 10 degrees of tilt, or even closer for thicker specimens or if higher resolution is required. Each image is digitized and Fourier transformed, and the data from all images are merged after they have been put on a common origin. The resulting 3D density map can be represented either as a balsa wood model (Fig. 9) or on a computer graphics screen (Figs 1 l(b), (d) and (g), 12(a-d), (g) and (h)).

Except for the limited resolution there are two major causes for concern about the reliability of 3D structures obtained from negatively stained specimens. One is the problem of the so-called missing cone. This refers to the missing cone of data in the 3D reconstructions, due to the fact that a complete 3D data set cannot be collected, because the specimen cannot be tilted up to 90 degrees. In most microscopes the goniometer stage allows tilting only up to 60 degrees. Tilt angles up to 90 degrees can be obtained by bending the grids before inserting them into the electron microscope, or by using special goniometers that allow full tilting of the specimen. Another way of adding in the data of the missing cone is to use sectioned samples. The effect of omitting the cone data are more serious for higher resolutions, and for the usual 2.5 nm resolution obtained with negatively stained specimens it is not very serious. The protein becomes blurred and slightly stretched in the direction perpendicular to the plane of the S-layer. An analysis of the effects of including limited tilt ranges can be found in Baumeister et al. (1987). The other problem, mentioned earlier, is that of the shrinkage of negative stain during exposure to electrons. After a long time of exposure the protein layer may shrink down to half its initial thickness. This can be reduced substantially by taking great care to reduce the radiation of the specimen, especially during focusing. In conclusion the 3D structures of S-layers can be relied on at the present level of resolution.

Three-dimensional reconstructions can also be made from metal shadowed specimens. A 3D structure determination of the S-layer of Deinococcus radiodurans by both metal shadowing and negatively stained specimens gave similar results, confirming the validity of both methods (Baumeister et al., 1986b).

IV. THREE-DIMENSIONAL STRUCTURES OF S-LAYERS

1. Classification of S-layers According to Symmetry

Two classification schemes for S-layers have been proposed. One scheme only distinguished between protein and pores, and based the classification on the presence of protein or pore at each of the symmetry elements in the unit cell (Sjrgren et al., 1985). Later Saxton and Baumeister (1986) proposed an improved classification scheme, in which they noted that the protein monomers in S-layers tend to be composed of one more massive (M) core and one lighter domain providing connectivity (C). They described the twelve different possible arrangements of two such domains with respect to the symmetry axes. The protein monomers were described as elongated molecules, with in every case only two sites of contact, namely the two ends of the protein. Most S-layers were found to have the M domain at the highest symmetry axis (Baumeister and Engelhardt, 1987). With the increased number of S-layer structures now determined, these schemes can be developed further, suggesting the new packing arrangements for S-layers with p4 and p6 symmetries, shown in Fig. 10.

Unlike the straight molecular models in the scheme of Saxton and Baumeister, the elongated protein monomers are often bent as a comma and have three sites of contact. In the tetragonal S-layers the protein masses at the two 4-folds have been found to face opposite sides of the S-layer.

The three possible points of contacts for each monomer are at each of the two ends and at the centre of the monomer, rather than only at the two ends as in the scheme of Saxton and Baumeister. The two most common plane groups for S-layers, p6 and p4 both contain 2-fold and higher symmetry axes. The central convex parts of the comma-shaped molecules may be expected to form contacts across 2-fold axes, but not across higher symmetry axes because of overcrowding. The ends, however, can form trimers, tetramers or hexamers.

JPB 5 1 : 2 - g

142 S. HO\,'M()LLER t't al.

FIG. 9. Balsa wood model of the 3D structure of the S-layer of Eubacterium sp. AHN 990, seen from three different directions. The two surfaces of the complicated structure are quite diffcrenl.


FIG. 9, Continued.

Interestingly enough, both in p4 and p6 there are twice as many possible high symmetry contacts {4- or 3- and 6-fold, respectively) as there are 2-fold contacts. This allows every monomer to have three contacts--a dimeric arrangement at the centre and higher symmetries at both ends of the molecule. In this way there will be only one basic type of arrangement for p4 and one for p6, as illustrated in Figs 10 and 11.

It seems that most tetragonal S-layers make full use of all the possible intermolecular contacts. In hexagonal S-layers it is more common to find only two out of the three possible kinds of contacts.

In the three possible space groups with lower symmetry, pl , p2 and p3, such an elaborate system of symmetric contacts is not possible, and this may be one reason why they are less commonly found in nature.

The symmetries of S-layers may depend on the composition of the underlying membrane or cell wall.

As can be seen from Table 1 in (Sleytr and Messner, 1983) about 60% of the S-layers on Gram-negative bacteria are hexagonal and only about 25% are tetragonal. For Gram- positive bacteria this relationship is nearly reversed. In Archaebacteria hexagonal symmetry is the most common. It is difficult to judge if these relationships are statistically significant, but they could be supported by the fact that both in Gram-negative bacteria and Archaebacteria, where the S-layer is attached to a lipid membrane, the hexagonal symmetry is preferred.

2. Hexagonal S-layers

The highest symmetry found for S-layers is p6, a hexagonal arrangement of six monomers per unit cell. This symmetry is the most commonly found for Archaebacteria and Gram- negative Eubacteria where the S-layers are in contact with an underlying lipid membrane. In Gram-positive bacteria hexagonal lattices are not so frequent although they do exist.

144 S. HOVMOLLER el al.

FI(~. 10. The arrangement of proteins in S-layers that arises when each protein monomer is described as an elongated, bent molecule, connecting to other monomers over all the symmetry axes. ta)

Tetragonal and (b) hexagonal packings.

The hexagonal S-layers have the largest unit cells, simply because there are more monomers in each hexagonal unit cell than is the case for the lower symmetries. The 3D structures of nine hexagonal S-layers have been determined until now, and their unit cell dimensions vary between 14 and 33 nm; whereas most tetragonal S-layers have unit cell dimensions under 13 nm. The area occupied per monomer is for most S-layers of any symmetry around 40 nm 2.

In the following description of the 3D structures of the individual S-layers, some basic data for every one is given in the first line, including the unit cell parameters a( = b) and thickness (c) in nanometers, the classification according to Saxton and Baumeister (1986) and the molecular weight (Mr)in kilodaltons (kDa), when available.

(a) Su!folobus acidocaldarius

Archaebacterium, p6, a = 22 nm, c = 10 n m , M 6 C 2 , M r 140-170 kDa.

The first S-layer structure to be elucidated in 3D was Sulfolobus acidocaldarius (Taylor et al., 1982; Deatherage et al., 1983). S. acidocaldarius lives in hot acidic springs. It has one of the largest and most complicated unit cells of all those whose 3D structures have yet been determined (Fig. 11 (a)). There are intermolecular contacts across both 6-, 3- and 2-fold axes. Many conformations of the polypeptide are consistent with the map. All tracings of the protein monomer favour an elongated molecule making a tight 3-fold contact on one side of the S-layer, a tight 2-fold contact near the centre of the S-layer and a more open ring-like contact around the 6-fold axis at the opposite side. Although the density map of the monomeric assembly is very clear, the stain does not penetrate between the monomers so as to separate them. That is the reason why several interpretations of the map are possible.

In spite of the complicated pattern of intermolecular contacts, the structure is quite


(a) (b)

(e) (d)

FIG. 11. Three-dimensional structures of hexagonal S-layers. (a) Sulfolobus acidocaldarius, from Amos et al., 1984. (b) Thermoproteus tenax, from Wildhaber and Baumeister, 1987. (c) Synechocystis sp. CLII, from Karlsson et al., 1983. (d) Clostridium thermohydrosulfuricum, from Cejka et al., 1986. (e) Deinococeus radiodurans, from Saxton et al., 1984. (f) Aquaspirillum serpens, from Dickson et al., 1986. (g) Acetoyeniurn kivui, from Baumeister and Engelhardt, 1987. (h) Bacteroides buccae, from

Sj6gren et al., 1985.

146 S. HO'VMf)LLIc.R et al.

(e) (t)

(g)

FK~. I I. C o n t i n u e d .

(h)


porous, with a large dome-shaped opening as seen from the cell side, only closed on the other side by a thin layer of protein, the ring around the 6-fold axis. From the molecular weight (140-170kDa), the volume of the protein in one unit cell can be estimated to be 6x 155,000x 1.25=1.1 x 106/~ 3 (assuming a protein density of 1.35g/cm 3 which is equivalent to a volume of 1.25/~3/Da). This is only about one quarter of the volume of the unit cell (220 x 220 x sin 60 x 100=4.2 x 106/~3).

(b) Thermoproteus tenax

Archaebacterium, p6, a = 33 nm, c = 6 nm (30 nm), M6C3, M r 325 kDa.

The sulphur-dependent extremely thermophilic Archaebacterium, Thermoproteus tenax, with a growth optimum near 90°C, has a remarkable S-layer. The 2D arrangement of this S-layer and that of the closely related T. neutrophilus were studied in negatively stained and freeze-dried and shadowed preparations by Messner et al. (1986). The hexagonal unit cells of these two species were quite similar, although not identical. The unit cell dimensions are the largest of all well-characterized S-layers. The main protein is found around the 6-fold axis. The suggested shape of the monomer is that of a very long molecule, first going radially out from the 6-fold axis, then after about 10 nm making a sharp bend, and via a dimeric contact after a further 15 nm ending in a trimeric contact. This S-layer is unusual in that it seems to have three intermolecular contacts which are not across crystal symmetry axes. This kind of contact is very rarely seen in other S-layers, and may only be possible because this monomer is so extended.

In the 3D structure determination of the S-layer of T. tenax (Wildhaber and Baumeister, 1987), the above arrangement was confirmed, but additional striking features were observed (Fig. 11 (b)). The structure as seen in the 2D projection turned out to be only about half of the S-layer architecture. The S-layer was described as a roof, resting on pillars. The pillars could of course not be determined in the 2D projection. The fundaments of the pillars rest on, or are embedded in, the plasma membrane. They are some 25-30 nm long, and support the perforated roof, which is about 6 nm thick.

Even in thin-sectioned cells the pillars are clearly seen, and one may speculate about what the purpose can be of the extracellular compartment this S-layer creates. Since the S-layer is only semipermeable, it has been suggested that the bacterium harbours distinct catabolic and transport enzymes here (Wildhaber and Baumeister, 1987). The roof of the S-layer would then allow free passage of solubilized molecules, while retaining larger proteins just outside the plasma membrane.

The S-layer in T. tenax is also exceptional in its rigidity. It retains the original cylindrical shape when freeze-dried, unlike many other S-layers that collapse into flat ghosts. T. tenax is a cylindrical rod, with an almost constant diameter of about 0.41/~m, while the length can vary between 1 and 80/zm. The S-layer consistently has one crystal axis very close to, but significantly off (by 3 4 degrees) from, the normal to the long axis of the rod-shaped bacteria. In this way the S-layer forms a right-handed two-strand helix with 38 + 3 unit cells per turn. The growing and dividing bacteria are always inside the protection of the S-layer. The S-layer is virtually free of lattice faults, except near the cell poles. A spiral arrangement of unit cells, unlike rings, may provide a possibility for the bacterium to grow by adding newly synthesized monomers to the S-layer at wedge dislocation, without completely upsetting the crystalline lattice.

(c) Synechocystis sp. CLII

Cyanobacterium, p6, a = 15.2 nm, c = 14 nm, M6C2, M r 100 kDa.

Cyanobacteria are oxygenic photosynthetic prokaryotes, formerly called blue-green algae. Several Cyanobacteria have been found to have S-layers (Kessel, 1978; Vaara, 1982), and the 3D structure of one of these has been determined, Synechocystis sp. CLII (Karlsson et al., 1983), Fig. ll(c).

This structure is more simple, with the main part of the protein arranged as a dense

148 S. HOVMOLLER et al.

hexamer around the 6-fold axis. Thin arms near the centre of the S-layer connect the hexamers via dimeric contacts at the 2-fold axes. There are large pores around the 3-fold axes of the structure. The unit cell dimensions are among the smallest found for hexagonal S-layers. There is a cavity in the centre of the hexamer, stretching from one side almost throughout the whole molecule.

(d) Eubacterium sp. AHN 990

Gram-positive, p6, a = 15.7 nm, c = 13 nm, M 6 C 2 .

The structure Eubacterium sp. AHN 990 shows the rather common arrangement of protein predominantly around the 6-fold axis (Sj6gren et al., 1988). This S-layer has been used for illustrating the various steps in a 3D structure determination throughout this review. The steps in the reconstruction can be followed from Fig. 4(b) with the negatively stained S-layer, via the digitized area (Fig. 6), the Fourier transform (Fig. 7), the 2D map before (Fig. 8(a)) and after imposing the exact crystallographic p6 symmetry (Fig. 8(b)) until the final 3D structure (Fig. 9).

There is a pore starting as a rather large opening (6.5 nm diameter) on one side of the S-layer around the 3-fold axis, splitting up into four smaller pores at the other side, with the largest opening (3 nm diameter) around the axis, and the other three pores symmetrically arranged around it. There are intermolecular contacts also around the 2-fold axes, so this is an example where the molecules associate across all three possible symmetry axes.

In projection (Fig. 8) the S-layer seems to consist of unrelated cog-wheel-like hexamers. The reason why the connections between monomers cannot be seen in projection is that they are mainly in a horizontal arrangement, and as such will not show up as strongly as a vertical column of protein when projected. This problem can only be overcome by 3D analysis of the structures.

(e ) CIostridium thermohydrosu!furicum

Gram-positive, p6, a = 16 nm, c = 9 n m , M 6 C 3 , M r 140 kDa.

Clostridium thermohydrosu!furicum grows at + 65°C and it has a glycoprotein forming a hexagonal S-layer. The structure follows the most common principle for hexagonal S-layers, with a massive core around the 6-fold axis, and the elongated monomers extending in a skewed manner, and meeting again at the opposite side of the S-layer, around the 3-fold axes (Cejka et al., 1986), Fig. ll(d).

It was possible to determine which side of the S-layer faces the cell and which faces the medium, by comparing the 3D model from negatively stained sheets with a surface relief from freeze-etching experiments. The part of the protein around the 6-fotd axis is facing the peptidoglycan layer of the cell.

The S-layer of Cl. thermohydrosu!furicum has a remarkable mechanical strength. S-layers isolated by mechanical disruption of cells, followed by lysozyme digestion of the peptidoglycan, existed almost exclusively as cylindrical sacculi. These would not even break by ultrasonication. As a consequence of this Cejka et al. could find only very small monolayers, and decided to determine the 3D structure from these double layers. There arc two problems with this strategy. One is the lower resolution commonly found for double layers, and the other is that the two layers will overlap in the images. While two similar layers superposed create a moire pattern which is very confusing for the naked eye, in the Fourier transform the two layers give rise to easily distinguished lattices of points. The data from one of the lattices can be extracted from the Fourier transform, and after inverse Fourier transformation reveals the structure of only that layer, without contributions from the other one. Cejka et al. applied an alternative technique for the separation of the data from the two layers of C1. thermohydrosu!furicum. They found the positions of the unit cells in real space by cross-correlation, and then averaged over some 300 unit cells in each layer. In this way they made two independent determinations (top and bottom layers) of the same structure. This provided a very good control of the reliability of the structure determination. The two reconstructed layers were very similar, in spite of the fact that only one was in contact with


the supporting carbon film, which might have been expected to lead to different staining of the two layers.

In an interesting molecular modelling experiment Woodcock et al. (1986) showed that the (putative) monomers of Cl. thermohydrosulfuricum and (the tetragonal) Cl. aceticum were very similar, and that the two layers could be transformed into each other after an extra domain in the CI. aceticum monomer had been removed.

(f) Deinococcus radiodurans

Gram-negative, p6, a = 18 nm, c=6.5 nm, M6C 2, M r 105 kDa.

The S-layer of the radio-tolerant bacterium Deinococcus radiodurans is one of the best studied from a structural point of view. The amino acid sequence has been determined (see Section VII), and the 3D structure has been studied by metal shadowing (Wildhaber et al., 1985), unstained freeze-dried preparations (Engel et al., 1982) and in negative stain (Baumeister et al., 1986b).

The S-layer is probably anchored to the underlying outer membrane by one or more hydrophobic sections of the polypeptide. On the outside the S-layer is covered by a carbohydrate coat.

The structure (Fig. 11 (e)) has a main density concentrated around the 6-fold axis, at the side facing the outer membrane. As in all S-layers with large protein masses around the 6-fold axes, there is a channel more or less through the whole hexamer, symmetrically arranged around the 6-fold axis. The real size of this channel is difficult to assess, and it should be pointed out that for obvious crystallographic reasons proteins must have an empty cylinder of some 0.5 nm in diameter around symmetry axes that are not screw axes. If an atom from one of the protein monomers would be on the axis, then all the other monomers would also have an atom at the very same position, which of course is physically impossible. Either there is some disorder very close to the symmetry axes, or there is an open channel. When the heavy metal salt is introduced as a staining material, it may enter this channel, and perhaps widen it further.

The main density of the hexamer is in the form of a ring with a radius of 2.2 nm. From this, six arms extend almost tangentially, further out making dimeric contacts with arms from the neighbouring hexamers. This provides the connectivity within the S-layer. The tips of the arms extend beyond the 2-fold axes, turning in towards the 3-folds, but not meeting to create a trimeric contact. Instead there is a rather large pore at the 3-fold position, with a radius of about 4 nm.

(g) Aquaspirrillum serpens

Gram-negative, p6, a = 14.5 nm, c = 6 (15)nm, M6C3, M r 140 kDa.

Aquaspirrillum serpens strain VHA has a hexagonal S-layer with a rather small unit cell (Dickson et al., 1986). Hexamers form a cup-shaped density, with no indications of an opening around the 6-fold axis (Fig. 1 l(f)). The putative monomers are Y-shaped, with the stem and one of the arms running close to and rather parallel to the 6-fold axes. The other arm stretches out towards the 3-fold axes, where three such arms meet in a trimeric contact. The thickness of this S-layer from the bottom of the hexagonal cup to the tips of the Y-linkers was estimated to 6 nm from the 3D structure determination. However other data, such as side-views, suggested a much thicker S-layer, up to 15.5 nm. There are elliptical pores of about 2.5 x 1.5 nm around the 2-fold axes.

The main body of the monomer would be an elongated protein molecule, with a distinct 2 nm long branch extending near the centre of the molecule. The branched, or Y-shaped appearance of the monomer is not uncommon among S-layer proteins, in contrast to most other proteins which tend to be rather more compact, either spherical or ellipsoidal.

(h) Acetogenium kivui

Gram-negative, p6, a = 19 nm, c=7 .6 nm, M6C3, M r 90 kDa.

Acetogenium kivui has an S-layer with a particularly clear domain structure (Fig. 11 (g)),

JPB 51: 2-~'

150 S. HOVM6LLER et al+

with six T-shaped heavy domains forming a funnel-shaped hexamer around the 6-fold axis (Baumeister and Engelhardt, 1987). The different hexamers are joined via thinner links, meeting at the 3-folds. The large opening of the funnel-shaped hexamer faces outwards.

(i) Bacteroides buccae

Gram-negative, p6, a=21.5 nm, c = 5 n m , M 3 C 2 , 6 , M r 70 kDa.

The thinnest and most delicate S-layer network found so far is that of Bacteroides buccae (Sj6gren et al., 1985). The veil-like S-layer is arranged in a chicken-wire-like net, which gives a transparent appearance in the electron microscope because of the many and large pores. Every unit cell has one round pore of 5 nm diameter around the 6-fold axis, and another six elongated pores of 5 x 2.5 nm (Fig. 1 l(h)). The tentatively assigned monomer stretches from the major mass at the 3-folds, over a dimeric contact at a 2-fold axis, turning towards the 6-fold axis, where it forms an open ring around the largest pore, together with five other monomers. The monomer is some 20 x 2 x 2 nm, with pronounced bends both in the plane and out of the plane. The apparent molecular weight estimated from SDS gel electrophoresis was 70 kDa (Kerosou et al., 1988).

B. buccae is one of several Bacteroides species which have been found in infected jawbones in humans. Sj6gren et al. found S-layers that were indistinguishable from that of B. buccae also in B. capillus and B. pentosaceus. Furthermore another crystalline protein layer with a much smaller unit cell (7.7 nm) was also found in all the strains. This may be interpreted as a further indication that these differently named Bacteroides species are in fact all the same species. An alternative possibility would be that the S-layers of these closely related strains has remained unchanged, at least at this level of resolution, after these bacteria diverged.

Bacteroides are Gram-negative bacteria, and as such they have both the cytoplasmic and an outer membrane. Both crystalline layers were found associated with the outer membrane, but in different manners. In thin-sectioned cells and in side views of negatively stained sheets (Fig. 4(a)), the 21.5 nm lattice was clearly seen to be outside (or above) the outer membrane. by as much as 20 nm. In contrast the 7.7 nm lattice was never seen extending outside the outer membrane, indicating that it was embedded in the lipid bilayer. This property together with the small unit cell dimension and the low molecular weight (17 kDa), indicate that this is a porin. Porins are frequent proteins in outer membranes of Gram-negative bacteria. They are not considered as proper S-layers.

The two different crystalline layers were usually found on separate sheets of membrane, but occasionally membranes with both crystal forms were found. In those cases the two crystals did not overlap, but they were in contact in the plane of the membrane. An explanation for this behaviour may be that the S-layer is anchored in the outer membrane. and that this anchor disturbs the formation of close-packed porins into a crystal. If this description is correct, then the S-layer of B. buccae would be quite similar to that of Thermoproteus tenax, with long narrow pillars anchored in the outer membrane and supporting a perforated roof. In the 3D reconstruction of B. buccae (Sj6gren et al., 1985) no such pillars were seen, but they may well be so thin and fragile that they are bent and disordered and therefore not visible in the averaged 3D model.

3. Tetragonal S-layers

The tetragonal arrangement is the most common for S-layers on Gram-positive Eubacteria but it is found also on other bacteria. The tetragonal S-layers have a more homogenous architecture than the hexagonal S-layers. Most have a major protein mass at one of the 4-folds, with the monomers radiating out from there, ending up in another tetrameric arrangement, usually on the opposite side of the S-layer. With the noticeable exception of the S-layer of Desulfurococcus mobilis all the p4 structures that have been determined in 3D fall in a very narrow range of unit cell dimensions, with a = h between 10.5 and 13 nm.


(a) Bacillus sphaericus

Gram-positive, p4, a = 13 nm, c = 8 nm, M4C2,4, M r 140 kDa.

Bacillus sphaericus P-l, earlier identified as a Bacillus brevis species, was one of the first S-layers to be studied in detail, although initially only as 2D projections by optical filtering (Aebi et al., 1974) then commonly called the T-layer (Tetragonal). It was also the first tetragonal S-layer for which a 3D structure determination was completed (Lepault et al., 1986) together with the very similar S-layer of the phylogenetically distantly related species Sporosarcina ureae (Engelhardt et al., 1986).

The S-layer can be obtained as large well-ordered sheets. In freeze-dried and metal shadowed preparations the two sides of the S-layer are very different. The side facing out of the bacterium is smooth, while that facing the underlying peptidoglycan layer appears rough.

The 3D structure (Fig. 12a) shows a major dome-shaped tetrameric domain on the side facing the bacterium. Each monomer has two arms extending out from the tetramer in opposite directions. Both arms stretch out towards the other kind of 4-fold axis. In this way there are eight rather than four monomers interacting at one single 4-fold, but this is possible since they are on different heights. The major arm goes further out from the peptidoglycan layer, and ends up in a compact tetramer on the outside of the S-layer.

(b) Sporosarcina ureae

Gram-positive, p4, a = 12.9 nm, c=6.8 nm, M4C2.4, M r 115 kDa.

The S-layer of Sporosarcina ureae (Engelhardt et al., 1986), Fig. 12(b), is very similar, not to say indistinguishable from that orB. sphaericus. The unit cell dimensions and the thickness do not differ significantly from those of the above mentioned B. sphaericus. The thicknesses of these two S-layers (6.8 and 8.0 nm) may seem significantly different, but the thickness is always less accurately determined than the unit cell dimensions in the plane of the S-layers for several reasons. Firstly there is the shrinkage of negatively stained specimens (see Section III.3), which has been found to affect only the thickness, not the in-plane distances. Secondly the protein molecules may be flattened upon contact with the grid, especially if there are some very thin protrusions on the surface. Thirdly, because of the missing cone of data corresponding to tilted views near 90 degrees, the data is of poorer quality in the third dimension. Finally there is the almost philosophical question of what is the thickness of a corrugated layer. In the plane the unit cell gives one unambiguous measure of the dimension, but with a thickness of only one unit cell, i.e. no repeating distance; this dimension is less easy to define. With these considerations in mind the S-layers of these two species are very similar.

For S. ureae there are also IR spectrum data available (Engelhardt et al., 1986), indicating a fl-structure composition of 37 + 5%.

(c) Clostridium aceticum

Gram-positive, p4, a= 12.1 nm, c=6.3 nm, M4C 4, M r 120 kDa.

The S-layer of Clostridium aceticum (Fig. 12(c)) is made up of cup-shaped tetramers, connecting via long arms around the other 4-fold axis (Woodcock et al., 1986). There is also in this species a minor arm, but unlike in B. sphaericus and S. ureae the minor arm of C1. aceticum is directed towards the 2-fold axis, and it seems to end blindly before meeting another monomer for contacts. It could not be assessed with absolute certainty which side of the S-layer faces the bacterium, but the appearance of metal shadowed preparations showing the two sides of the layer supported the suggestion that the major mass centre is on the inner surface, as has been found for many other S-layers (Baumeister and Engelhardt, 1986).

The distinct cup-shape of this S-layer is strikingly similar to the cup-shaped major protein mass of CI. thermohydrosulfuricum. This is especially interesting because the latter S-layer is hexagonal, so it may be seen as an indication of a close relation of S-layer proteins even with different symmetries. Woodcock et al. (1986) showed that if the minor side lobe of the monomer from Cl. aceticum was cut off, the remaining protein mass could be arranged to form an S-layer not only similar in shape but also in lattice constant (16 nm) with that of

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(9) (h)

FIG. 12. Three-dimensional structures of tetragonal S-layers. (a) Bacillus sphaericus, from Lepault et al., 1986. {b) Sporosarcina ureae, from Engelhardt et al., 1986. (c) Clostridium aceticum, from Woodcock et al., 1986. (d) Clostridium thermosaccharolyticum, from Cejka and Baumeister, 1987. (e) Euhacterium sp. ES4C, from Sj6gren et al., 1988. (f) Aeromonas hydrophila, from A1-Karadaghi et al., 1988. (g) Azotobacter vinelandi, from Bingle et al., 1987. (h) Desulfurococcus mobilis, from Wildhaber

et al., 1987.

Cl. thermohydrosulfuricum. With sequence data and genetic information available in the future it will be possible to prove if this modelling is biologically relevant.

(d) Clostridium thermosaccharolyticum

Gram-positive, p4, a = 11.0 nm, c = 6.9 nm, M4C 4.

The 2D projection of the S-layer of Clostridium thermosaccharolyticum is quite similar to that of CI. aceticum, yet there are distinct differences in their 3D strucutres (Cejka and Baumeister, 1987), Fig. 12(d). The unit cell of Cl. aceticum is slightly larger in the plane, but on the other hand not as thick as that of Cl. thermosaccharolyticum. In the 3D projection of Cl. thermosaccharolyticum the protein core around the 4-fold is more dominant, and the connecting arms appear weaker.

(e) Eubacterium sp. ES4C

Gram-positive, p4, a = 10.6 nm, c=9 .5 nm, M4C2, 4.

The S-layer of Eubacterium sp. ES4C is very evident even in negatively stained whole bacteria. The in-plane dimensions are the smallest among the tetragonal S-layers whose 3D structures have been elucidated. The structure (Sj6gren et al., 1988) follows the common pattern for p4 S-layers, with a major protein mass arranged as a tetramer on one side of the membrane, and a minor mass around the other 4-fold axis protruding on the opposite side. This arrangement becomes very clear in a side-view, as seen in Fig. 12(e).

(f) Aeromonas hydrophila

Gram-negative, p4, a = 12 nm, c = 8.4 nm, M4C 4, M r 52 kDa.

A eromonas hydrophila is a pathogenic bacterium for fish and mammals, including humans. Recently it has been shown that the S-layer most probably is responsible for the virulence (Dooley and Trust, 1988).

The 3D structure of A. hydrophila (A1-Karadaghi et al., 1988) shows the common tetragonal arrangement with one major tetramer at one side of the S-layer and a minor tetramer at the opposite side (Fig. 12(f)). The monomer stretches from one tetramer to the

154 S. HOVM6LLER et al.

other, and half-way in between it makes a further dimeric contact across the two-fold axis. The dimensions of this tentative monomer are 2 × 2.5 × 13 rim.

The closely related bacterium A. salmonicida is an important fish pathogen, and it has been shown that its S-layer is essential for the virulence (Ishiguro et al., 1981).

The molecular weight (49 kDa), unit cell dimensions (11.5 to 12.0 nm) and 2D projection of A. salmonicida (Stewart et al., 1986) are very similar to those of A. hydrophila, strongly suggesting that these S-layers are very similar.

(g) Azotobacter vinelandi

Gram-negative, p4, a = 12.5 nm, c = 5 nm, M4C 4, M r 60 kDa.

Azotobacter vinelandi has an S-layer which is similar to those of Aeromonas mentioned above with two large domains, one at each of the two 4-fold axes (Bingle et al., 1987). In all these three S-layers one of the tetramers is star-shaped while the other looks more compact. From the 2D projection of the S-layer of A. vinelandi there seems to be very large S-shaped pores, 3.5 nm wide and 11 nm long. The 3D map (Fig. 12(g)) shows some weak densities crossing these large pores, and it is likely that some very fine protein structures of about I nm diameter cross the S-shaped pores near the centre of the S-layer. This would considerably decrease the size of the pores.

(h) Desulfurococcus mobilis

Archaebacterium, p4, a = 18 nm, c = 6 nm, M4C 2, M r 50 kDa.

The extreme thermophilic Archaebacterium, Desuljurococcus mobilis, lives in soifataric hot springs of temperatures up to 97°C, at acidic pH. Its S-layer looks distinctly different from the ones described above. The 3D structure was determined by Wildhaber et al. (1987). The unit cell area is about twice as large as any of the other tetragonal S-layers described above. Yet the molecular weight of the monomer is among the smaller of the proteins forming tetragonal S-layers. Thus the network formed by this filamentous protein is a very open one, with cross- shaped pores of 12 × 12 nm (Fig. 12(h)). There is a small possibility that some very fine polypeptide chains, invisible at the 2 nm resolution limit, cross or partly occlude these large pores. If not, the pores would allow free passage of very large molecules, up to about 700 kDa.

Unlike many other tetragonal S-layers, that of D. mobilis makes only one tetragonal contact, and additional contacts over the 2-fold axes, leaving large pores at the remaining 4-fold axes. The tetrameric contacts seem to be stronger than the dimeric ones, since isolated small fragments of the S-layer show star-shaped features with four arms, resembling the tetramers in the S-layer.

As in all Archaebacteria the S-layer of D. mobilis is immediately outside the plasma membrane. It is likely that the S-layer is anchored to the membrane at the 4-folds, where the protein protrudes from the plane of the S-layer towards the cell side.

Another unusual property of this S-layer is its very high degree of lattice disorder. This may be an effect of the spherical cell of D. mobilis. Cylindrical bacteria can and frequently do have perfect lattices except at the cell poles, but a perfect lattice is not compatible with a spherical shape.

4. Oblique S-layers

(a) Methanospirillum hungatei

Archaebacterium, pl, a=2 .9 nm, b= 12.0 nm, 7--93.7, c=3 .5 nm.

The methanogenic A rchaebaeterium, Methanospirillum hungatei, has an extremely unusual cell wall architecture. Several bacteria share a crystalline sheath, and together they form long filaments. The individual cells inside are separated by spacer elements, called septa. Both the cell sheaths and the septa are crystalline arrangements of protein. The 3D structures of both have been determined by Shaw et al. (1985).


The septa are hexagonal cylindrical plates with a uniform diameter of about 0.4 #m. The unit cell dimension are 18.2 nm, and the protein is mainly arranged around the 3-fold axes, leaving very large pores (15 nm) around the 6-folds. Each pair of cells are separated by two septa, having the same polarity, i.e. front to back rather than back to back.

The sheath may be considered as an S-layer, but it is remarkably different in many aspects. The symmetry appears only to be oblique although every unit cell contains four, probably identical, beadlike subunits. The monomers are about 3 × 3 x 3 nm. Four subunits lie on a row, making together a unit cell of 2.9 x 12.0 nm, with a thickness of 3--4 nm. Three of the subunits appear very similar, while the fourth is smaller and more ellipsoidal than the others. The long unit cell axis is parallel to the axis of the filaments. Thus the structural units are arranged as cylindrical rather than helical bands.

The normal way for bacteria to enclose the cell by an S-layer is by introducing lattice faults which close off the S-layer at the ends. In M. hungatei, rather than forming a single type of crystal lattice, this organism has adopted the strategy to produce one cylindrical surface and closing off the ends with fiat discs of completely different composition.

V. REGULAR OUTER MEMBRANE PROTEINS

In Gram-negative bacteria there are two lipid bilayers in the cell envelope; the cytoplasmic membrane and the outer membrane, as illustrated in Fig. 2. The cytoplasmic membrane is very complex, with a large number of different proteins inserted into the lipid bilayer. Bacteria do not have any internal membranes as eukaryotic cells do. All the membrane proteins they need for transport of nutrients into the cells, discarding waste products, keeping the salt balance in the cell and for respiration and/or photosynthesis are located in the cytoplasmic membrane. The outer membrane is much simpler, and only contains a few different proteins. When many copies of a single kind of protein are present in a small volume they often crystallize. This happens also in the outer membrane of bacteria. These proteins are called regular outer membrane proteins (rOMP), and are considered distinct from the proper S-layer proteins.

1. Porins

The most common proteins in outer membranes of Gram-negative bacteria are the porins. It is also a main protein of the mitochondrial outer membrane, supporting the hypothesis that mitochindria originate from a primitive aerobic bacterium that was taken up by an anaerobic bacterium. These two bacteria continued to live in symbiosis and may be the origin to eukaryotes.

Porins are smaller than the S-layer monomers, with molecular weights of about 30 kDa. They form channels through which water soluble molecules up to about 600 Da diffuse, while excluding larger molecules (Nikaido and Vaara, 1985). In Escherichia coli there are four distinct porins, all forming trimers.

The structure of porins has been studied both by electron microscopy (Dorset et al., 1983; Mannella and Frank, 1984; Lepault et al., 1988) and by X-ray diffraction (Garavito et al., 1983). Porins commonly form crystals with p3 symmetry in vivo, with unit cells around 8 nm. Porin was one of the two first membrane proteins to be purified and crystallized as 3D crystals, large enough to diffract X-rays. The crystals diffract to 2.7 A resolution, but unfortunately it has proven difficult to find good heavy atom derivatives, so the atomic structure is still not elucidated.

2. Other Regular Outer Membrane Proteins

In the outer membrane of Chlamydia trachomatis there is a larger protein (40 kDa) that forms hexagonal arrays (Chang et al., 1982) and resembles in many ways hexagonal S-layers. It has unit cell dimensions a--b = 17.5 nm and is 15 nm thick. The structure is a complex network of protein with a pore or large depression around the 6-fold axis.

Various strains of Pseudomonas acidovarans have been reported to possess regular arrays of protein molecules. A 3D structure determination of one of these showed a compact arrangement of subunits in a p4 lattice with unit cell dimensions a = b = 11 nm, c--- 5 nm

156 S. HOVMC~LLER et al.

(Chalcroft et al., 1986). The relatively low molecular weight (32 kDa) and the fact that the outer membrane could not be removed without destroying the crystalline order of the proteins, indicate that this is an rOMP and not an S-layer.

VI. OTHER C R Y S T A L L I N E SHEETS OF COAT P R O T E I N S

Bacterial envelopes are not unique in nature in showing a 2D crystalline array of proteins. On the contrary there are many such crystals, in a variety of organisms and cells. Whenever a membrane is filled mainly by one protein and this protein is present in high concentration, it is likely to form crystals. This is the case for specialized membranes involved in photosynthesis, like photosynthetic reaction centres in Rhodopseudomonas viridis (Michel, 1982; Deisenhofer et al., 1984) and bacteriorhodopsin in Halobacterium halobium (Henderson and Unwin, 1975). These proteins are membrane bound with specific enzymatic reactivities, and as such are not believed to be closely related to S-layer proteins, However, there are also cases of proteins forming coats on viruses, algae, bacterial spores and cells of higher organisms, and it may well happen that some of these in the future will be shown to be related to the S-layer proteins.

1. Bacterial Spore Coats

Bacterial spores are highly organized, densely packed and stable structures. They can withstand much harsher conditions than the active bacteria, such as high temperatures or total dryness. Not surprisingly they are surrounded by a rigid and highly complex wall.

In the case of Bacteroides thuringiensis (Ebersold et al., 1981 ) as many as ten different kinds of membranes or other layers have been identified in the spore wall, which in total is about 300 nm thick. Several of the different kinds of layers are themselves multilayers. The laminar layer is composed of about ten identically looking layers. These are all crystalline, as are also the pitted layer and the cross-patched layers of the spore coat and the so-called crystalline layer of the outermost component of the spore, the exosporium. The different crystalline layers all seem to be hexagonal, with unit cell dimensions in the range from 5 to 10 nm, typical for a regularly ordered protein. In summary a bacterial spore may contain some 20 to 30 layers of at least 10 different types, with about half of these being crystalline protein layers.

2. Algal Cell Walls

Algae are eukaryotic cells, and as such are much more complex than bacteria. Crystalline cell wall layers have been observed in some green algae belonging to the group of Volvocales (Roberts et al., 1982). Two-dimensional crystals of glycoproteins were found as part of the cell walls of Chlorogonium elongatum, Chlamydomonas asymmetrica, Chlamydomonas reinhardii and Labomonas piriformis. Large sheets of well-ordered crystals could be observed by electron microscopy of negatively stained cell walls. The unit cell dimensions of these crystals varied from 21 x 7--20 x 38 nm. Unlike the bacterial S-layers which almost all have high symmetries (p4 or p6) the algal cell walls were all found to be oblique with space group p2.

Because of uneven staining or distortions of the crystal lattice, the symmetry observed in negatively stained crystals may often be lower than the true one. However, if this is the case for some of the algal cell walls, this would lead to even more striking differences between algal and bacterial surface layers. The p2 lattices found may in some cases in reality have p22121 symmetry. Indications of such higher symmetry can be seen in Chlorogonium elongatum where metal shadowed images show a doubling of the unit cell compared to that found in the negatively stained cell walls. This was interpreted as "corrugation of the rows, where alternate subunits are slightly raised" (Roberts et al., 1982). This may be the effect of alternate molecules facing different ways in the cell wall. Such arrangements have never been observed for bacterial S-layers.

A 3D structure of the crystalline layer of Labomonas pirijbrmis (Shaw and Hills, 1982) showed both similarities and differences when compared to bacterial S-layers. The crystals had unit cell dimensions a = 24.7 nm, b = 16.7 nm, 7 = 109° and a thickness of 7-8 nm. The


symmetry was oblique, p2. The monomers were elongated, with the long axis more than three times longer than the short axes. There were pores also through this crystalline lattice.

3. Viral Coats

Viruses are much simpler and smaller organisms than bacteria. The smallest viruses are not much more than a protein coat that protects the genetic material that codes for the coat. In small helical and spherical viruses the only proteins in the virus particle are in fact the coat proteins. These coats are the only protection for the viral RNA or DNA, and they are very densely packed, in a crystalline way. With the need to form a much smaller closed body, some 30 nm compared to several hundred nm for bacteria, virus have evolved other ways of crystallization. In spherical viruses the symmetry is icosahedral, involving 5-fold symmetry. This is because it is the most efficient way of forming a small closed body (Caspar and Klug, 1962). One helical plant virus, tobacco mosaic virus (Bloomer et al., 1978) and several spherical viruses, including common cold (Rossmann et al., 1985) and polio virus (Hogle et al., 1985) have now been determined by X-ray diffraction to atomic resolution. A review article by Liljas (1986) is recommended.

One of the most significant, and perhaps surprising results of these 3D structure determinations has been the remarkable similarity between the foldings of the coat proteins of all the small spherical viruses. Although the sequences were not obviously related and the habitats of for example polio virus and tomato bushy stunt virus are very remote, the 3D folding of the polypeptide chains are very similar. Thus it is likely that many if not all small spherical viruses have evolved from a common ancestor.

Viral coat proteins are cone-shaped and make a very tight fit, impermeable even to water, whereas the S-layers are porous structures.

Larger spherical viruses, like adenovirus dilute the pentameric proteins with hexameric proteins (hexons) in order to increase the volume. The hexons of adenovirus, when isolated can form a 2D crystal, in many ways resembling an S-layer (van Oostrum et al., 1986). The size of the hexon is also similar to that of S-layer proteins, with 967 amino acids in the single polypeptide. The hexon structure has been determined to atomic resolution by X-ray crystallography on 3D crystals (Burnett et al., 1985).

4. Gap Junctions

Multicellular organisms are dependent on communication between cells. This can be carried out in a variety of ways, and one of these is via gap junctions. Gap junctions are proteins forming a 2D hexagonal crystal on each of two adjacent cells. The two crystals are in register, and together they form a highly specific channel, allowing small molecules to pass from one cell to its neighbour without spilling into the surrounding medium. A similar function as connections has been proposed for S-layers by Baumeister and Hegerl (1986). Gap junction crystals have also been studied in 3D by electron microscopy (Unwin and Ennis, 1984).

VII. AMINO ACID SEQUENCES OF S-LAYER PROTEINS

Apart from 3D structure determinations, the other essential information needed for an understanding of the architecture of S-layers is amino acid sequences. With the rapid development of DNA-sequencing of genes, we are just at the beginning of a very exciting era with a dramatic increase in the number of sequences available. Until 1985 very little sequence data on S-layer were published, namely short N-terminal fragments from some different strains ofAeromonas salmonicida (Kay et al., 1984). Today we have already the full sequences of at least three S-layer proteins, including pieces of the DNA around the gene itself. Thereby we can start to compare S-layers from different species in great detail, and to understand the molecular biology of the mechanisms of promotion and export through the bacterial membrane(s) of these proteins.

The S-layer protein at the surface of the bacteria is expected to be a major antigen and also a recognition site for bacteriophages. As such it is reasonable to expect a rapid mutation rate for these proteins in order to evade such attacks.


Amino acid compositions from many different S-layers show a common scheme of acidic proteins. In most bacteria the S-layer is a single polypeptide of some 400 to 2000 residues.

1. Homologies in Closely Related Strain,s

Kay et al. (1984) compared the 27 first N-terminal amino acids from Aeromonas salmonicida in three different species. As may be expected the amino acid sequences of these species were highly conserved with 23 out of 27 amino acids identical. The S-layer of A. salmonicida is probably the best studied from a functional point of view. This Gram- negative bacterium causes the lethal disease furuncolosis in a variety of fish species, such as Canadian trout, gold-fish and salmon etc. Mutants devoid of S-layers lose their virulence. In the acute form of this disease, bacteria grow rapidly in the major body organs, producing a terminal septicaemia often accompanied by severe tissue necrosis.

2. Sequences of Distantly Related Bacteria

The complete genes coding for the S-layer polypeptides have been determined for the two S-layers, the outer (OWP) and middle wall-protein (MWP), in Bacillus bret;is 47 (Tsuboi et al., 1986, 1988) and for the single S-layer in Deinococcus radiodurans (Peters et al., 1987). The genes coding for S-layers in Caulobacter crescentus (Smit and Agabian, 1984) and Aeromonas salmonicida (Belland and Trust, 1987) have also been cloned.

The proteins forming the two S-layers of B. brevis and that of D. radiodurans are all about 1000 amino acids long. Without the help of 3D structure determinations, comparisons of these sequences have only showed one stretch of amino acids with a slightly significant homology. This is a stretch of 76 amino acids around 200 amino acids from the C-terminal end, 13 out of which were identical and 45 conservative replacements, when the sequences of D. radiodurans and the OWP ofB. brevis were compared (Peters et al., 1987). Near the centre of this stretch there was a sequence of five consecutive identical amino acids, Lys-Arg-Asn- Aia-Ser. In the same region of the MWP ofB. brevis there is also a similar sequence; Lys-Lys- Lys-Ala-Ser. When more sequences are available we will be able to determine whether this is a significant feature of S-layers or if the similarity is only fortuitous.

The two S-layer proteins from B. brevis have a larger net negative charge than that of D. radiodurans. The OWP and MWP have a high concentration of net negative charges in the C-terminal part of the proteins, with over hatf the net negative charges in the last third of the proteins. Also in D. radiodurans the charges are accumulating in the C-terminal end, but without the pronounced overweight of negative charges.

In order to anchor a protein in a lipid bilayer an alpha helix of about 25 purely hydrophobic amino acids is needed. In D. radiodurans there are five stretches of 20 or more uncharged amino acids, including one of 43 amino acids. This S-layer shows strong interaction with the underlying outer membrane and it is likely that the hydrophobic stretches found in the sequence are forming alpha helices that transverse the membrane. In B. brevis neither of the two S-layers show such long hydrophobic stretches. This fits well with the fact that both the MWP and the OWP are shed from the bacterial medium and accumulate in the medium at the stationary phase of bacterial growth (Yamagata et al., 1987). Since B. brevis is a Gram-positive bacterium it does not have an outer membrane to anchor an S-layer in.

3. Signal Sequences

The S-layers are synthesized in the cytoplasm of the bacteria and then exported through one (Gram-positive) or two (Gram-negative) lipid bilayers to reach their destination on the outside of the cell envelopes. The transport of proteins across lipid bilayers is known to be mediated by specific proteins spanning the membranes. Proteins that are to be exported carry a special leader or signal sequence, which is split off during the process of passing the membrane. This signal sequence immediately precedes the N-terminal start of the protein. Every organism has its specific signal sequences, but there are also common features. Typical for signal sequences are two lysines at the N-terminal end of the signal, followed by about 20 hydrophobic amino acids (yon Heijne, 1985). All the three S-layer sequences mentioned


above do indeed have such signal sequences. The signal sequences of the two S-layer proteins from B. brevis show a strong homology with 13 identities out of 23 amino acids.

4. Prediction of 3D Structures from Amino Acid Sequences

The amino acid sequence determines the folding of a protein. In a given environment (pH, temperature etc.) the folding of a protein will always be the same. Since all cells have to be able to cope with at least some variation in chemical composition during their lifetime, it is essential that the protein folding remains the same over a reasonable range of chemical and physical conditions. These facts are encouraging in the sense that we may be able to predict the 3D structures of proteins just from the (now relatively easily available) amino acid sequences, without the need to carry out the major undertaking of an X-ray diffraction study of the crystalline protein. However, although Nature knows how to fold the proteins, we do not. The reason is the immense difficulty of the problem. Even with the sequence given, there are two angles of rotational freedom for every amino acid in the polypeptide backbone, plus the rotations of the side groups. For a protein with hundreds of amino acids the number of different possible foldings becomes astronomical. There are several methods of trying to solve the problem, using both theoretical calculations and semi-empirical data (Jones and Thirup, 1986) but the problem of predicting a complete 3D folding of a protein of which no near relative has yet been solved by X-ray diffraction is still unsolved.

Before the first S-layer structures are solved to atomic resolution by X-ray crystallography, we can only make some good guesses about some features, such as the prediction of hydrophobic stretches that anchor the S-layer in outer membranes of Gram-negative bacteria, but a reliable modelling to atomic resolution is not possible.

VIII. BIOLOGICAL FUNCTIONS OF S-LAYERS

With the abundant presence of S-layers on bacteria, it is clear that these structures are vital for many organisms. There are experimental evidence for functions such as protection against a hostile environment, specific adhesion, interaction with bacteriophages and shape determination. Furthermore, it should be emphasized that S-layers are always an integral part of the total bacterial envelope, both structurally and functionally. Therefore the function of S-layers may not be found by just considering the S-layer itself, disregarding the interactions with the other components of the cell envelope.

1. Protection against Hostile Environment

S-layers are found with very high frequency on bacteria living in an extreme environment, such as high temperature or high salt concentrations. This suggests that the S-layer may have an important role in protecting the cell from a hostile environment.

In a study of different strains of Bacillus stearothermophilus, Messner et al. (1984) found S-layers on 30 out of the 39 strains investigated. This is a lower limit, since S-layers may go undetected if they are disrupted during the preparation procedure for electron microscopy, or if they are hidden by polysaccharides. It is also possible that some strains had S-layers in their natural habitat, but lost them under the non-competitive laboratory conditions. S-layers have also been found on Sulfolobus acidocaldariu.s (Deatherage et al., 1983) which lives in hot acidic springs, Clostridium thermohydrosulfuricum (Cejka et al., 1986) and on many halophilic bacteria (Sleytr and Messner, 1983).

It may well be that most bacteria living under such extreme conditions have S-layers, but how these S-layers can protect against the environment is not understood yet. In fact all the B. stearothermophilus S-layers mentioned above were perfectly penetrable by the protein lysozyme, proving that all these S-layers must have pores larger than 3.5 nm wide.

2. Specific Adhesion

The surface properties of the whole bacteria, including the charges, are largely determined by the S-layers. They determine the ability to adhere to specific cells (Sleytr and Messner. 1983). In Aeromonas salmonicida it has been shown that the S-layers are needed for the virulence of the bacteria (Evenberg and Lugtenberg, 1982).

160 s. HOVM6LLER et al.

3. Interaction with Bacteriophages

With an S-layer as the outermost feature of a bacterium it is expected to be a major recognition site (commonly called receptor) for bacteriophages. Indeed this has also been observed in several cases.

Bacillus sphaericus P-1 has a tetragonal S-layer as described above [Section IV.3(a)], which has been found to act as a bacteriophage receptor (Howard and Tipper, 1973). A virulent bacteriophage was isolated from soil. It was inactivated by purified S-layer. Twenty- four phage-resistant mutants were isolated, all of which still had S-layers, although many with apparently smaller molecular weights. The mutants did not absorb phage, and the cell walls from the mutants did not inactivate the phages.

The S-layer of Aeromonas salmonicida is lost if bacteria are grown at temperatures above 25~C. The bacteria devoid of S-layer show an increased sensitivity to bacteriophage (Ishiguro et al., 1981). In this case the S-layer serves a protective function.

4. Shape Determinin9 Coat

Since isolated S-layers have the ability to assemble into cylinders or closed vesicles, it was speculated that the S-layers were responsible for determining the shape of the bacteria. However, mutants without S-layers have been found to have the same shape as the wild-type, for both Gram-positive and Gram-negative bacteria. Therefore it is considered unlikely that S-layers have a shape-determining function in these bacteria (Sleytr and Messner, 1983). On the other hand it has been confirmed that S-layers in some cases are needed for maintaining a rod shape in Archaebacteria such as Halobacteria where no rigid peptidoglycan layer is present.

IX. B I O T E C H N O L O G I C A L A P P L I C A T I O N S

The S-layers have special properties that may be exploited in biotechnological applications. Because of their very fine and accurate mesh, S-layers may be used as filters. S-layers are produced in very high amounts and exported out of the cell and this may also capture the interest of biotechnologists.

1. Ultrafiltration Membranes

An ideal filter should fulfil many criteria. One of the most important is a constant pore size: other criteria are high pore density and chemical stability. S-layers form just such filters--they have an exactly defined pore size, a porosity of 20-70% and an extremely high pore density (5 x 1012/cm2).

Sieytr and Sfira have investigated the properties of S-layers for ultrafiltration purposes (Sleytr and Sfira, 1986; Sfira and Sleytr, 1987a,b). They extracted S-layers from bacteria and deposited the intact sheets on microfilters with a pore size of 100 nm. The S-layers were treated with glutaraldehyde to improve the chemical and mechanical stability of the membranes. These filters showed a very sharp cut-off in the size of molecules that could pass through the filters. The cut-offs are different for different bacterial strains, depending on the S-layer structures and pore sizes. For this reason an S-layer ultrafilter with desired pore size may be found for any specification in the range I 6 nm. For example the S-layers from the strain Bacillus stearothermophilus NRS 1536/3c allowed 95-100% of the protein carbonic anhydrase (M r 30 kDa) to pass through while only 10-15% of the ovalbumin (M r 43 kDa) could pass through the filter. The high accuracy of these filters may be even more appreciated when the diameters of these proteins are compared--ovalbumin is only 12% wider than carbonic anhydrase (assuming spherical shapes). A protein with twice the diameter of these, ferritin, was used to check the integrity of the S-layers. Ferritin is about the size of a single unit cell of the S-layer. The ferritin was rejected to 100% which showed that the membranes were intact.

The possible uses of S-layer ultrafiltration membranes for commercial and scientific applications are presently being investigated, and the idea has been patented (Sleytr and S/ira, 1984).


2. Genetic Engineering

The facts that S-layers are produced in large amounts and exported out of the bacterial cells may be taken advantage of in genetic engineering applications. It may be possible to use the promotor of the S-layer monomers for other genes that are cloned, so that these are also produced in very large quantities. Furthermore the signal sequences of S-layers may be coupled to the N-terminal end of proteins that are cloned in these bacteria, and in this way the proteins would be exported out of the cells. There they would be easy to harvest, and they would not inhibit the life of the bacteria by accumulating inside the cells. The possible prospects of such applications may be appreciated by the ability of Bacillus brevis to secrete as much as 12 grams of protein per litre under optimal conditions (Tsukagoshi et al., 1984). The protein secreted consists mainly of the two S-layers, the OWP and MWP proteins.

X. CONCLUDING REMARKS

The crystalline surface layers of bacteria are important for many reasons. Their abundance in nature shows that they fulfil vital functions for the bacteria. Our lack of understanding of the functions of S-layers should not lead us to think they may not have any, but rather prompt us to further investigate why it is so important for many bacteria to produce these S-layers. In some cases, notably for the Aeromonas species the S-layers are important for the virulence, and they may be used in the development of vaccines. Several of the unusual features of S-layers may also be taken advantage of for commercial purposes, in biotechnological applications.

The advance of our knowledge of the structures and properties of S-layers will hopefully answer many of the imminent questions in the near future. With the advent of atomic resolution structures, the amino acid sequences will become more interpretable from a structural point of view. By comparing structures and sequences of S-layers we have one more tool to investigate relationships between bacteria. With data on mutation sites in the S-layers we will be able to enter into the field of dynamic interactions on a molecular level between different organisms, in this case between bacteria and attacking bacteriophages which use S-layers as recognition sites.

ACKNOWLEDGMENTS

The authors wish to express their thanks to all the colleagues in the field of S-layers for sharing results and ideas, and especially to Professor Uwe Sleytr and his group for arranging the workshops on S-layers in Vienna. This work has been financially supported by grants from the Swedish Natural Sciences Research Council (NFR) and Sven och Ebba-Christina Hagbergs Stiftelse.

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