Characterization of the ultrastructure and the self-assembly of the surface layer of Bacillus stearothermophilus strain NRS 2004/3a

JOURNAL OF ULTRASTRUCTURE AND MOLECULAR STRUCTURE RESEARCH 97, 73-88 (1986)

Characterization of the Ultrastructure and the Self-Assembly of the Surface Layer of Bacillus stearothermophilus

Strain NRS 2004/3a

PAUL MESSNER, DIETMAR PUM, AND U W E B. SLEYTR

Zentrum J'fir Ultrastrukturforschung und Ludwig Boltzmann Institut f~r Ultrastrukturforschung, Universitiit fiir Bodenkultur, A- 1180 Wien, Austria

Received January 21, 1987, and in revised form May 12, 1987

The ultrastructure of the crystalline surface layer (S-layer) of Bacillus stearothermophilus strain NRS 2004/3a has been characterized by electron microscopy supplemented by optical and computer image analysis. The S-layer, composed of glycoprotein subunits, has oblique symmetry, and can be extracted by guanidine hydroehloride. Upon dialysis, this extract produced both flat and cylindrical mono- and double-layer self-assembly products. Optical diffraction analysis of negatively stained preparations showed five types of double-layered assembly products. Computer filtering separated the double-layer complexes and revealed them to be composed of a common monolayer with p2-symmetry (a = 9.4 nm, b = 11.6 nm, and 3' = ca. 78°). By analysis of freeze-dried and heavy metal-shadowed self-assemblies the surface topography and the characteristic "handedness" of the morphological units have been determined. Labeling with polycationic ferritin has shown that each surface of the S-layer possessed a different net charge. The results indicate that S-layers in vivo could prevent autoagglutination of cells. © 1986 Academic Press, Inc.

Many eubacteria and archaebacteria pos- sess crystalline arrays of subunits as the out- ermost component of the cell envelope (Sleytr, 1978; Beveridge, 1981; Sleytr and Messner, 1983; Sleytr et al., 1986a). The majority of these surface layers (S-layers) are formed from a single protein or glycoprotein which is strain specific with molecular weights from 40 000 to 200 000. S-layers generally cover the entire cell surface and may exhibit hexagonal, square, or oblique lattices with center-to-center spacings of the morphological units varying from ca. 5 to 35 nm.

Various methods have been developed for their isolation and purification (Sleytr and Messner, 1983; Koval and Murray, 1984), and frequently in vitro self-assembly of isolated S-layer subunits into flat sheets, open ended cylinders, or closed vesicles can be induced without additional wall compo- nents (Sleytr, 1981; Sleytr and Messner, 1983).

In analogy to other bacterial cell surface structures, S-layers most probably have evolved as a consequence of interactions be-

73

tween the cells and their environment. It can be assumed that S-layers have the po- tential to function as barriers against exter- nal or internal factors, as promotors for cell adhesion and surface recognition, and as a supporting framework involved in deter- mining and maintaining the shape of cells which have no rigid wall component (Sleytr and Messner, 1983; Messner et al., 1986a).

Previous chemical analyses of the puff- fled S-layer material obtained from the strain described in this study have shown that the crystalline array is composed of glycoprotein subunits (Kiipcii et aL, 1984; Christian et al., 1986; Sleytr et aL, 1986b; Messner et al., 1987). This paper describes the ultrastructure of the S-layer of Bacillus stearothermophilus NRS 2004/3a in detail to a nominal resolution of 1.9 nm by using electron microscopy and computer image processing.

MATERIALS AND METHODS

Preparation of the S-Layers

B. stearothermophilus, strain NRS 2004/3a, was grown in continuous culture (Messner et aL, 1984). The

0889-1605/86 $3.00 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

74 MESSNER, PUM, AND SLEYTR

S-layer glycoprotein was obtained from cell wall preparations by extraction with 5 M guanidine hydrochloride (puriss.p.a.; Fluka, Buchs, Switzerland)(Sleytr and Glauert, 1976). To produce different assembly products the extract was then thoroughly dialyzed at 60°C against several changes of either distilled water or solutions of low (2.5-10 raM) or high (50-500 mM) concentrations of CaC12, MgC12, NaCI, or LiC1 in water, respectively, containing 0.01% NaN3 to inhibit micro- bial contamination. The self-assembly products formed during the dialysis were chemically characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described (Messner et aL, 1984) and then the remaining product was stored at 4°C. During dialysis, the time-dependent decrease of the guanidine hydrochloride concentration in the dialysis bags was monitored by electrical conductivity reduction of the solution using a WTW LF 521 conductivity meter (Wissenschaftlich-Technische Werk- st~itten, Weilheim i.Ob., FRG).

Preparative SDS--PAGE was performed with a BRL PG1101 preparative gel electrophoresis system (Be- thesda Research Laboratories, Gaithersburg, MD) with a 4% stacking gel (1 cm gel height) and a 6% separation gel (3 cm gel height) as described in the instruction manual. SDS was removed from the pooled solution by precipitation with acetone according to Hager and Burgess (1980). The precipitate was resuspended in 5 M guanidine hydrochloride and after dialysis against distilled water the assembly products were stored at 4°C.

Electron Microscopy and Specimen Preparation

Freeze-etching of intact cells was carried out in a Leybold BIOETCH 2005 (Leybold Heraeus, Cologne, FRG) freeze-etching unit (Sleytr and Umrath, 1976). Fracturing and etching were done at -95°C and the samples were etched for 2 min. Shadowing with plat- inum-carbon (Pt/C) and cleaning of the replica was described previously (Messner et aL, 1984).

Negative staining was performed as described (Mess- ner et al., 1986a). After adsorption of the protein assemblies to the grid they were crosslinked on one drop of 2.5% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.2) for 20 rain. This step was necessary because self- assembly structures have been shown to disintegrate in a broad spectrum of negative staining solutions without prefixation (Messner et aL, 1984). Subsequently the samples were stained with uranyl acetate (Messner et al., 1986a).

Labeling of self-assembly products with polycationic ferritin (PCF) (Sigma Chemical Co., Munich, FRG) was performed as previously described (Sleytr and Friers, 1978; Messner et aL, 1986a). These preparations were then fixed in glutaraldehyde and negatively stained as mentioned above.

Native and PCF-labeled self-assembly products were freeze-dried at -80°C according to Messner et al.

(1986a). The dried samples were shadowed at 450C either with Pt/C (1.0 nm) or tantalum-tungsten (TaJ W, 0.5 nm) and examined in the electron microscope immediately after preparation.

The adsorption of the S-layer preparations onto positively charged surfaces was studied by using grids coated with Alcian blue. Formvar- and carbon-coated grids were floated for 30 sec on one drop of a 0.1% Alcian blue solution (Serva, Heidelberg, FRG) and then washed by passage over two drops of distilled water. After air- drying, these grids were used for both negative staining and freeze-drying experiments as described.

Specimens were examined in a Philips EM 301 electron microscope at nominal magnification of x 45 000 (Messner et aL, 1986a).

Image Processing

Fourier domain processing and correlation averaging were used on the image processing part as described previously (Messner et aL, 1986a). The regularity of the p2-symmetry was assessed by computing the phase residual comparing p 1 and p2 structure factors (Baker et aL, 1985). Amplitudes and phases on positions common to both reciprocal lattices were restored as described by Misell (1978). "One-sided" filtering of superimposed lattices was used to obtain single-layer averages because the structural preservation of the double layers was better than that of monolayers. A multivariate two-sample t test was used to test the signifi- cance of differences within and between lower and upper layer structure factors (BMDP Statistical Software 1981, option Hotelling T 2, Department of Biomathematics, University of California, Los Angeles).

The characteristic folding angles (c~ and a') of curved and collapsed double-layer assembly products were determined from the corresponding diffraction pattern of negatively stained preparations. The folding line of the structure on the micrograph was aligned parallel to the sharp, opaque edge of a rectangular mask and the angle was measured from the mirror line between the short reciprocal base vectors to the line of reflection maxima from the edge.

RESULTS

L a t t i c e O r i e n t a t i o n on the I n t a c t Ce l l

F r e e z e - e t c h e d p r e p a r a t i o n s o f B. s tearo-

t h e r m o p h i l u s N R S 2 0 0 4 / 3 a s h o w e d a h i g h l y

o r d e r e d o b l i q u e S - l a y e r l a t t i c e w h i c h c o v -

e r e d t h e ce l l s c o m p l e t e l y (Fig . 1). O n t h e

c y l i n d r i c a l p a r t o f m o s t b a c t e r i a , t h e o b l i q u e

l a t t i c e h a d a n o r i e n t a t i o n w i t h t h e s h o r t l a t -

t i c e v e c t o r a a p p r o x i m a t e l y p a r a l l e l t o t h e

l o n g i t u d i n a l a x i s o f t h e cell . T h e h e m i s p h e r -

i c a l c a p s o n t h e ce l l p o l e s w e r e c o v e r e d b y

a d j a c e n t , r a n d o m l y o r i e n t e d S - l a y e r c r y s -

SURFACE LAYER ULTRASTRUCTURE AND SELF-ASSEMBLY 75

FIG. 1. Part of the cell envelope of a freeze-etched and Pt/C-shadowed cell of Bacillus stearothermophilus NRS 2004/3a. Arrows indicate the base vectors of the oblique S-layer lattice. Bar = 100 nm.

FIr. 2. Cell pole of a freeze-etched B. Stearothermophilus cell exhibiting crystal boundaries in the S-layer lattice (arrows). Bar = 100 nm.

FIG. 3. Cell pole of a freeze-etched B. stearotherrnophilus cell showing lattice distortions (arrows). Bar = 100 nm.

tallites. Crystal boundar ies (Fig. 2) and lattice dis tor t ions (Fig. 3) were clearly visible.

Characterization o f the S-Layer Material and Self-Assembly Experiments

The S-layer subunits could be isolated f rom the cell wall prepara t ions by 5 M gua- n id ine h y d r o c h l o r i d e ex t rac t ions . S D S - P A G E gels revealed four distinct bands (Fig. 4) w i th a p p a r e n t m o l e c u l a r we igh t s o f 93 000, 120 000, 147 000, and 170 000. Re- m o v a l o f the chaotropic agent by dialysis at 60°C (Sleytr and Glauert , 1976) yielded characterist ic self-assembly products which

appeared within 15 m i n after the start o f the dialysis (Jaenicke et al., 1985). Within 10 min the guanidine hydrochlor ide concentra t ion decreased f rom 5 to ca. 1 M and after 1 hr less than 0.01 M w a s left. A lattice ne twork on self-assembly products could only be visualized by negat ive stain after it was chemical ly fixed with 2.5% glutaraldehyde. Unfixed assemblies were feature- less.

Depending on the salt concentra t ion during dialysis and dialysis durat ion, different self-assembly structures were formed. In the presence o f the ions, listed in Table I, after


( k D ) a b

1 7 0 ~ ..... . 1 4 7 ~ ~ " 1 2 0 ~ ~ '~ ..........

9 3 ~ ~

® a.U

®

® m

~ ~ . ~ 2 ~ . ~ . ~ ,<~ ~,~ . -~-~ ~

SURFACE LAYER ULTRASTRUCTURE AND SELF-ASSEMBLY

TABLE I DIALYSIS CONDITIONS AND SELF-ASSEMBLY PRODUCTS OF S-LAYER SUBUNITS

77

Cation Low concentration (2.5-10 mM) High concentration (50-500 mM)

Ca 2+ Large, medium, and small cylinders; sheets

Mg 2+ Large, medium, and small cylinders; sheets

Na + Small cylinders; sheets

Li + Small, morphologically not well-defined structures; a few small cylinders

Predominantly small and medium cylinders; sheets

Predominantly small and medium cylinders; sheets

Small, morphologically not well-defined structures

Unstructured precipitations

30 m i n m a i n l y c y l i n d r i c a l s e l f - a s s e m b l y p r o d u c t s w i t h d i a m e t e r s b e t w e e n ca. 70 a n d 220 n m were f o r m e d (Fig. 5). U p o n p r o - l o n g e d d i a l y s i s (48 hr) i n c l u d i n g seve ra l changes o f the d i a l y s i s m e d i u m (Tab l e I) the f o r m a t i o n o f large c y l i n d e r s w i th d i a m e t e r s o f ca. 1 /~m a n d o f shee t s w i t h a r eas o f u p to 10 t tm 2 (Figs. 6, 7) c o u l d b e o b s e r v e d . G e n e r a l l y , t he p re sence o f l ow c o n c e n t r a - t i o n s o f b i v a l e n t c a t i o n s w i t h i n 48 h r l ed to the f o r m a t i o n o f a m i x t u r e o f w e l l - d e f i n e d shee ts a n d c y l i n d r i c a l a s s e m b l y p r o d u c t s . S e l f - a s s e m b l y p r o d u c t s o b t a i n e d b y d i a l y s i s aga in s t MgC12 were , o n ave rage , s l igh t ly less r egu la r ly o r d e r e d t h a n t h o s e o b t a i n e d w i t h CaC12. A s a c o n s e q u e n c e , for h igh r e s o l u t i o n s tud ie s i n c l u d i n g c o m p u t e r i m a g e r econ - s t r u c t i o n p r o c e d u r e s o n l y s e l f - a s s e m b l y s t ruc tu re s o b t a i n e d in the p r e s e n c e o f C a 2+ i o n s were used .

H i g h c o n c e n t r a t i o n s o f CaC12 o r t he

p r e s e n c e o f m o n o v a l e n t c a t i o n s in low con- c e n t r a t i o n s in the d i a l y s i s m e d i a y i e l d e d p r a c t i c a l l y o n l y c y l i n d r i c a l s e l f - a s s e m b l y p r o d u c t s (Tab l e I) w i t h d i a m e t e r s o f ca. 130 n m . L i t h i u m ions s e e m e d to i n h i b i t the as- s e m b l y p roc e s s a n d a t h igh c o n c e n t r a t i o n s o n l y s t r u c t u r e l e s s p r e c i p i t a t i o n s o f t h e S - l a y e r p r o t e i n occu r r ed . C h a n g i n g the d i - a lys is m e d i u m to a so lu t i on o f 2.5 m M CaC12 for 24 h r a t 60°C a l l o w e d the c o m p l e t e r ea r - r a n g e m e n t o f these p r e c i p i t a t e s in to h igh ly o r d e r e d a s s e m b l y p r o d u c t s , i d e n t i c a l to t h o s e o b t a i n e d u p o n d i r e c t d i a l y s i s o f the g u a n i d i n e h y d r o c h l o r i d e e x t r a c t a ga in s t l ow CaC12 c o n c e n t r a t i o n s . C o n v e r s e l y , c y l i n d r i - cal a n d shee t - l i ke s t ruc tu re s w h i c h were f o r m e d b y d i a ly s i s a ga in s t l ow c o n c e n t r a - t i o n s o f CaC12 c h a n g e d i n to u n s t r u c t u r e d p r e c i p i t a t i o n s u p o n t r ans fe r i n t o 500 m M LiC1 so lu t ions .

S e p a r a t i o n o f the fou r d i s t i n c t S - l aye r

FIG. 4. SDS-polyacrylamide gel electrophoresis analysis of (a) the SDS-soluble whole cell extract of B. stearothermophilus NRS 2004/3a and (b) the purified S-layer glycoprotein after guanidine hydrochloride extraction.

Fio. 5. A mixture of large, medium, and small sized cylindrical S-layer self-assembly products obtained by dialysis of the guanidine hydrochloride extract against low concentrations of CaC12 at 60°C. The self-assembly products are negatively stained with 1% uranyl acetate. Bar = 1 ~m.

FIG. 6. Upon prolonged incubation at 60°C in CaC12 less small sized cylinders, but increasing numbers of sheets are observed. Bar = 1 urn.

FIG. 7. Negatively stained images of sheet-like assembly products obtained upon dialysis against a low concentration of CaC12. The assembly products exhibit different Moir6 patterns and characteristic folding angles. Bar = 100 nm.

FIG, 8. Negatively stained self-assembly products obtained upon dialysis of the 120-kDa protein band isolated by preparative SDS-polyacrylamide gel electrophoresis. Bar = 200 nm.

FIO. 9. Pt/C-shadowed S-layer composed of randomly oriented crystallites as obtained upon attachment to a carbon layer positively charged with Alcian blue. The primitive base vectors show the same handedness as on the intact bacteria. Bar = 50 nm.

78 MESSNER, PUM,

protein bands seen on the SDS-electrophoresis gel (Fig. 4) was possible by preparative SDS-PAGE. After removal of the detergent each of the individual protein bands reas- sembled into self-assembly products identical to the aggregates formed upon dialysis of a mixture of all four bands (Fig. 8 is rep- resentative). Additionally, small sized cylinders with diameters of ca. 30 nm were observed.

High Resolution Studies on Negatively Stained Self-Assembly Products

Evaluat ion of the negatively stained S-layer self-assembly products obtained un- der different dialysis conditions (Table I) revealed significant differences. Most of the small cylinders consisted of monolayers which showed a different stain intensity in comparison to the large and medium size cylinders and flat sheets. They were composed of double layers. The small cylinders were found in two populations differing in their diameters (ca. 70 and 100 nm) and their lattice orientation in relation to the longitudinal axis of the cylinder. By optical diffraction analysis it was found that the orientation of the two longitudinal axes dif- fered by 90 ° (Fig. 27).

In some negatively stained preparations small monolayer crystallites were observed. Pretreatment of the grids with Alcian blue increased the amount of adsorbed monolayer assembly products. Despite the" crazy paving appearance (Fig. 9), all crystallites exhibited an identical handedness of the base vectors of the p2-1attice (Henry and Lons- dale, 1969), which was identical to that observed on intact cells (Figs. 1-3).

Optical diffraction analysis of negatively stained images of cylindrical and sheet-like double-layer assembly products allowed superimposition assessment of the two layers involved. Five different Molt6 patterns were observed, and the corresponding diffraction patterns and lattice orientations are sum- marized in Fig. 10.

All double layers revealed a back-to-back orientation of the constituent monolayers

AND SLEYTR

and, with the exception of type I, all other types showed a characteristic lateral displacement of the layers (Fig. 10). In type I both layers had short base vectors in common and only sheet-like assembly structures were found. In type II the two layers were oriented along the long base vectors of the oblique lattice but staggered by one half repeat along the short base vector with respect to each other. Type II double layers could form both cylindrical (av diameters 1000, 570, and 220 nm) and sheet-like assembly products (Table I and Fig. 10). Type III was found in sheets and in small sized cylinders (av diameter 130 nm) (Fig. 11). The two layers were oriented along the (2,

- 1) lattice lines but were displaced by half the perpendicular distance between successive lines. Because this type shared only two common reflections [(2, - 1), (4, -2)] in the reciprocal lattice within the resolution limit it was predominantly used for deriving the structure of the monolayers by computer filtering. In type IV the two layers were oriented along the short diagonals of the oblique unit cell (1, - 1 ) lattice lines but were displaced by half the perpendicular distance between successive (1, - 1) lattice lines. This type formed only sheets. Type V also formed only sheetsand the two layers involved were oriented along the long diagonals of the unit cell [(1, 1) lattice lines] and were displaced buha l f the perpendicular distance between successive (1, 1) lattice lines.

Type II was found to be the most abun- dant type ofassembty products, followed by type IV and type III. Type I and V double layers were only rarely detected.

Exact measurements of the lattice parameters revealed significant differences among different assembly products (Fig. 10). While the length of the base vectors of the lattice (a = 9.4 _+ 0.2 nm and b = 1 1 . 6 _ 0 . 3 n m ) did not change in all self-assembly products, the interaxial angle seemed to be type-specific. In types I and II, a mean value of 78.3 ° was.found, while in types III, IV, and V the mean was 81.1 °. Both monolayer cylinders showed a mean interaxial angle of 84.0 °.


"One-sided filtering" of negatively stained double-layer assembly products of type III (Figs. 11, 12) showed no significant differences in the stain exclusion patterns of both the reconstructed upper (Fig. 13) and lower (Fig. 14) layer. This is based on the result of the multivariate two-sample t test. To evaluate the structural reliability of the reconstructions of the two layers involved in type III the one-sided filtering was repeated with negatively stained double layers of type II (Fig. 15) and type IV (not shown). For image reconstruction of type II, reflections common to both single layers (Fig. 16) were obtained from the corresponding values of type III. As already found in type III, the reconstructions of the upper (Fig. 17) and lower (Fig. 18) layers were compatible and revealed a similar staining behavior. The high regularity of the p2-1attice which be- came evident by comparing p l- (Figs. 13, 14, 17, 18) and p2-averages (not shown) could be quantified from the phase residual values (7-8 ° ) for all reconstructions up to the resolution limit. The averaged two-dimensional projections of the monolayers

showed well-defined stain/protein boundaries with sharp gradients of density. The bulk of the protein was in close vicinity to the central twofold axis of the unit cell and displayed a characteristic handedness (e.g., Fig. 13). Large stain-filled cavities ran parallel to the short base vector of the p2-1at- tice.

Characterization of the S-Layer Surface Structure by Freeze-Drying

Freeze-dried and heavy metal-shadowed cell wall preparations of B. stearothermophilus NRS 2004/3a with known orientation in the microscope were used to characterize the handedness of the morphological units and primitive base vectors of the S-layer lattice (Sleytr et al., 1982). Both Pt/C (Figs. 19, 20) and Ta/W (Figs. 21, 22) shadowed preparations revealed a resolution of structural details down to ca. 2 nm, as determined by the radial correlation function (Saxton and Baumeister, 1982). Quite similar images of the surface topography were obtained with both shadowing materials but the reconstructions of the Ta/

FIG. 10. Comparative analysis of the mono- and double-layer self-assembly products. (A) Schematic drawings of the monolayer lattice and the five types of double-layers including lateral displacements. The two layers are discriminated in the schemes by either full lines and filled circles or dashed lines and open circles. (B) Electron micrographs of type-specific self-assembly products exhibiting the characteristic folding angles (a and a') of the collapsed structures. The micrographs of the monolayer assembly products reveal both (a) the 70 nm and Co) the 100 nm cylinder. (C) Schematic drawings of the corresponding diffraction patterns. The superimposed lattices are represented by full or dotted lines. Filled circles represent the diffraction spots. (D) Lattice parameters of the assembly products; a and b, short and long base vectors (nm); 3' interaxial angle; a and a' folding angles of the collapsed structures.

FIG. 11. Negatively stained image of a type III double-layer assembly product. Bar = 100 nm. FIG. 12. Corresponding diffraction pattern to Fig. 11. Bar = 1/~ nm-t . FIG. 13. Computer image reconstruction of p 1 averages of the upper layer of a negatively stained type III

double-layer assembly product. The size and orientation of the unit cell are outlined and represent the best fit to the lattices shown in Figs. 19-22. Bar = 20 nm.

FIG. 14. Computer image reconstruction of p l averages of the lower layer of a negatively stained type III double-layer assembly product. The reconstruction is displayed mirror-symmetrically to facilitate the comparison with Fig, 13. Bar = 20 rim.

FIG. 15. Negatively stained image of a type II double-layer assembly product. Bar = 100 nm. FIG. 16. Corresponding diffraction pattern to Fig. 15. Bar = 1/~ nm-t . FIG. 17. Computer image reconstruction of p 1 averages of the upper layer of a negatively stained type II

double-layer assembly product. Bar = 20 nm. FIG. 18. Computer image reconstruction of p 1 average of the lower layer of a negatively stained type II

double-layer assembly product. The reconstruction is displayed mirror-symmetrically to facilitate the comparison with Fig. 17. Bar = 20 rim.

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Fro. 19. Computer image reconstruction of a freeze-dried and Pt/C-shadowed S-layer self-assembly product. The size and orientation of the unit cell are outlined and represent the best fit to the lattice shown in Fig. 13. The direction of shadowing is indicated by the arrow. Bar = 20 nm.

F~o. 20. Computer image reconstruction of a Pt/C-shadowed S-layer self-assembly product similar to Fig. 19, but the direction of shadowing differs by ca. 90 °. Bar = 20 nm.

FIG. 21. Computer image reconstruction of a freeze-dried and Ta/W shadowed S-layer self-assembly product. The shadowing direction is indicated by the arrow. Bar = 20 nm.

Fx~. 22. Computer image reconstruction of a Ta/W shadowed S-layer self-assembly product similar to Fig. 21, but the direction of shadowing differs by ca. 90 °. Bar = 20 nm.

W - s h a d o w e d a s s e m b l y p r o d u c t s e x h i b i t e d

m o r e c o m p l e x surface detai ls .

Labeling with Polycationic Ferritin

T o d e m o n s t r a t e poss ib le differences in the

charge d i s t r i b u t i o n o f S- layer surfaces o f in-

tact cells and se l f - a s sembly p roduc t s , b o t h

p r e p a r a t i o n s were l abe led wi th P C F a n d

subsequen t ly eva lua t ed by f reeze-dry ing and

h e a v y m e t a l s h a d o w i n g or by nega t i ve

s ta ining. In a c c o r d a n c e w i t h ear l ie r obser -

v a t i o n s (Sleytr a n d Friers , 1978) in tac t cells

il SURFACE LAYER ULTRASTRUCTURE AND SELF-ASSEMBLY 83

FIG. 23. Negatively stained image of the 70 and 100 nm monolayer cylinders. PCF is only bound to the inner surface of the cylinders. Bar = 100 nm.

FIG. 24. Freeze-dried and Pt/C-shadowed 70 nm monolayer cylinder labeled with PCF as in Fig. 23. Bar = 100 nm.

F~G. 25. Negatively stained image of a medium size double-layer cylinder and a double-layer sheet labeled with PCF. The marker molecules did bind only at areas where monolayers are exposed (asterisk). Bar = 200 nm.

FIG. 26. Freeze-dried and Pt/C-shadowed preparation as in Fig. 25. Bar = 200 nm.

o f B. stearothermophilus N R S 2 0 0 4 / 3 a d i d

n o t b i n d P C F to t h e S - l a y e r su r f ace . B o t h

t y p e s o f m o n o l a y e r c y l i n d e r s d i d b i n d t h e

p o s i t i v e l y c h a r g e d m a r k e r m o l e c u l e s o n t h e

i n n e r s u r f a c e (Figs . 23 , 24). O n t h e o t h e r

h a n d n o t a l l t y p e s o f d o u b l e - l a y e r c y l i n d e r s


longitudinal /- \ o loo

, PCF "% d ~ ~ "--..C_urvat ure of

Ty (dour

axis of the monolayer cylinder

the monolayer cylinder

'~ 70 nm

PCF m o

Q.

Y'I A B

B A

IIIIIIIII1

possible directions of curvatures of double- layers

FIG. 27. Schematic representation of the formation of mono- and double-layer assembly products. On the monolayer sheet A the axes of 70 and 100 nm@ monolayer cylinders are indicated. One of the axes includes an angle of 24 ° to the short base vector of the S-layer lattice. The second axis is perpendicular to the first. Both monolayer cylinders have an identical direction of curvature. Due to differences in the charge distribution on the S-layer surfaces polycationic ferritin (PCF) is only bound to the inner surface of both types of monolayer cylinders. Five types of double-layer assembly products with back-to-back orientation of the constituent monolayers have been found. The superimposition of sheets A and B for the double-layer assembly products of type I is demonstrated and the angular displacement of sheet B with respect to A around point X for the assembly products of type II to V is indicated (compare with Fig. 10 which also shows the lateral displacement of both


and the double-layer sheets could be labeled with the marker. PCF-binding only oc- curred on the open ends of the cylinders or on sheets where small areas of monolayers were exposed (Figs. 25, 26). These findings clearly demonstrated that differences in the net charge of both S-layer surfaces exist and that in all double-layer self-assembly products the two S-layers are oriented in such a way that the surfaces exposed are identical to the S-layer surface seen on intact cells (Fig. 27).

DISCUSSION

S-layers could be demonstrated on the surface of many Bacillaceae including Ba- cillus (e.g., Nermut and Murray, 1967; Holt and Leadbetter, 1969; Henry, 1972; How- ard and Tipper, 1973; Tsuboi et al., 1982; Abe et al., 1983; Abe and Kimoto, 1984; Messner et al., 1984; Doyle et al., 1986), Clostridium (e.g., Hollaus and Sleytr, 1972; Sleytr and Glauert, 1976; Kawata et al., 1984), Desulfotornaculum (Sleytr et aL, 1986b), and Sporosarcina (Beveridge, 1979) species. So far, all high resolution studies on Bacillaceae have been performed on square or hexagonal lattices (e.g., Aebi et al., 1973; Crowther and Sleytr, 1977; Stew- art and Beveridge, 1980; Burley and Mur- ray, 1983).

The only high resolution 2-D projection map of a p2-1attice has so far been reported for Aquaspiril lum putridiconchylium (Stew- art et al., 1980).

Isolated S-layer subunits from different Bacillaceae have shown the ability to assemble into regular arrays with the same lattice dimensions as those observed on intact cells (Aebi et aL, 1973; Sleytr, 1976; Hastie and Brinton, 1979a, 1979b; Sleytr and Plohber- ger, 1980; Sleytr, 1981; Sleytr et al., 1986b). Since in oblique lattices the orientation of

the two-dimensional crystal can be easily determined by the handedness of the lattice base vectors we were able to analyze the assembly route leading to the differently shaped mono- or double-layer self-assembly structures. S-layer glycoproteins extracted from cell walls of B. stearothermophilus NRS 2004/3a by high molar concentrations of guanidine hydrochloride have shown upon dialysis the ability to aggregate into a broad spectrum of differently shaped self- assembly structures (Table I). The ability of isolated S-layer subunits to assemble into fiat sheets or cylindrical structures was previously described for the constituent subunits of the p4-1attice on Bacillus sphaericus (Aebi et aL, 1973; Hastie and Brinton, 1979a). These authors explained the structural change from a fiat into a cylindrical self-assembly structure by a proteolytic cleavage of the S-layer protein which had been demonstrated by SDS-PAGE.

In our studies we have compared by SDS- PAGE the molecular weight distribution of the S-layer subunits in SDS extracts of whole cells with that of different S-layer preparations (e.g., early and late stages during the dialysis procedures) but could not find any detectable changes in the characteristic pattern of bands (Fig. 4). We therefore con- cluded that mechanisms other than proteolytic cleavage were responsible for the formation of differently shaped mono- and double-layer self-assembly products.

Two types of monolayer cylinders have been found with statistically significant different diameters. The lattice base vectors of both types of monolayer cylinders revealed the same handedness but a different orientation in relation to the longitudinal axes of the cylinder. We explain this phenomenon by the ability of the S-layer protomers to generate monolayers which have the inher-

lattices involved). As the monolayers have the inherent inclination to curve along two axes, cylindrical or flat double-layer assembly products are possible, depending on the degree of neutralization of the "internal bending strain."

86 MESSNER, PUM,

ent inclination to curve along two distinct axes into the same direction (Fig. 27). The different radii of curvature of the cylinders thus reflect the two low free energy states of the monolayer in which there is least residual stress between the constituent glycoprotein subunits.

On intact cells S-layers cover a cylindrical surface with a curvature considerably larger than both curvatures observed in monolayer self-assembly cylinders. Because S-layer lattices on the cylindrical part of intact cells (Fig. 1) have approximately the same orientation as observed on the 100- nm monolayer cylinder we assume that this in vivo alignment also reflects a situation in which there is a minimum of distortion stress within the lattice. S-layers on intact cells and S-layers generated on, or adsorbed to, positively charged surfaces (e.g., surfaces treated with Alcian blue) reveal the same handedness of the base vectors (Fig. 9). Fur- ther labeling expriments with PCF have shown that the S-layer has a net negative charge on one surface and this must be the surface which binds to the Alcian blue/grid surface. Thus, for the attachment of S-layers to positively charged surfaces, electrostatic interactions seem to be responsible. On the other hand, as the peptidoglycan layer of Bacillaceae (including B. stearothermophilus NRS 2004/3a) has shown the ability for binding PCF (Sleytr and Friers, 1978; Sleytr and Plohberger, 1980; Sonnenfeld et al., 1985) it is likely that bivalent cations form necessary salt bridges between the negatively charged surfaces of the peptidoglycan and the S-layers. Differences in the binding strength of S-layers to negatively and positively charged surfaces are reflected by the long range order of the lattices. Whereas the cylindrical part of the cells is generally cov- ered by large well-aligned S-layer lattices (Fig. 1), S-layers adsorbed to positively charged surfaces are fragmented into small randomly oriented crystallites (Sleytr and Glauert, 1975) (Fig. 9). This crazy paving appearance indicates that S-layer subunits adhering to positively charged surfaces have

AND SLEYTR

less mobility for recrystallization than un- der in vivo conditions.

The ionic conditions prevailing during the dialysis of the S-layer extracts determine the structure o f the double-layer assembly products. High concentrations of bivalent ions favored the formation of the cylindrical assembly products with small diameters. The structural organization of double-layer assembly products could be destroyed re- versibly by high concentrations of monovalent ions (Table I). We assume that by substitution the bivalent ions involved in stabilizing the double layers by salt bridges between the negatively charged groups can be replaced by monovalent ions and vice versa.

All self-assemblies composed of double layers have been shown to consist of two back-to-back oriented monolayer leaflets. This was deducible from the following experiments: (i) all metal-shadowed flat or cylindrical self-assembly structures revealed the same handedness of the primitive base vectors of the lattice, and (ii) PCF was bound to only one side of the monolayers (inside of the monolayer cylinders) but neither double layers nor intact bacteria were labeled (Sleytr et al., 1982), It was found that in double layers the characteristic alignment of the monolayers with respect to each other determines the shape of the self-assembly product (Figs. 10, 27). Because each of the back-to-back oriented monolayers has the intrinsic tendency to curve over two axes (as reflected by the two types of monolayer cylinders), frequently more than one type of self-assembly structure is found for each type of double layer (Figs. 5-7). Thus, it must be determined at a very early stage of the assembly process whether flat sheets or cylinders are formed. It is likely that the path- way for one of the various possible free- energy arrangements is determined when only a few morphological units have aligned themselves and that differences in the growth rates of the two monolayers involved are of significant importance. A complete neutralization of the "internal bending strain"

SURFACE LAYER ULTRASTRUCTURE AND SELF-ASSEMBLY

in the two monolayers seems to occur only in type I lattice where exclusively flat sheets are observed. The difference of approximately 3 ° in the interaxial angles between either type I and II or III, IV, and V of the double-layer assembly products is not yet fully understood; in both groups cylinders as well as fiat sheets are formed (Fig. 10). A possible explanation for these inconsis- tencies could be lattice distortions generated by linkages between the two monolayers.

The double-layer structures have been shown to collapse along type-specific folding angles as seen in freeze-dried or negative staining preparation (Figs. 7, 10). The orientation of the base vectors in the collapsed structures are statistically significant and can be used to determine the direction of curvature of the self-assembly products in sus- pension.

The observation that isolated S-layers have a strong tendency to associate with their inner faces reflects the in vivo situation, since intact cells of B. stearothermophilus NRS 2004/3a exhibit no tendency to aggregate in culture. Recently, this observation has been confirmed with another B. stearothermophilus strain (Sfira and Sleytr, 1987).

Because S-layer material separates into four distinct bands by preparative SDS- PAGE (seen in Fig. 4) and all lead to identical assembly products (Fig. 8), we con- clude that the morphogenetic information for the different assembly structures resides exclusively in the protein moiety and is not dependent on the amount of attached carbohydrate. The biological function of the carbohydrate residues previously characterized (Christian et al., 1986; Messner et aL, 1986b; Messner et al., 1987) is not yet fully understood, but investigations are in pro- gress.

We thank Dr. Terry Beveridge for critical reading of the manuscript and Miss Claudia Hurban for skilful technical assistance. This work was supported by grants from the "0sterreichischer Fonds zur Frrderung der wissenschaftlichen Forschung," Projects 4613 and 5290 and the"Osterreichisches Bundesministerium t'fir Wis- senschaft und Forschung."

87

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Documents

Characterization of the ultrastructure and the self-assembly of the surface layer of Bacillus stearothermophilus strain NRS 2004/3a