JOURNAL OF BACTERIOLOGY, Sept. 1987, p. 4092-4098 Vol. 169, No. 90021-9193/87/094092-07$02.00/0Copyright © 1987, American Society for Microbiology

Molecular Sieving through S Layers ofBacillus stearothermophilus Strains

MARGIT SARA* AND UWE B. SLEYTR

Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann-Institut far Ultrastrukturforschung, Universitat furBodenkultur, A-1180 Vienna, Austria

Received 30 March 1987/Accepted 10 June 1987

The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selectedstrains of BaciUus stearothermophilus were investigated. Measurements were performed of passive soluteuptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were preparedfrom whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preservedthe integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found toconsist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete Slayer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi thepeptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selectedfor the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The Slayers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showedsharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter ofabout 4.5 nm.

Many species of eubacteria and archaebacteria possess acrystalline surface layer (S layer) as the outermost compo-nent of their cell envelope (for reviews, see references 27through 29). S layers are composed of protein or glycopro-tein subunits which form porous meshworks with poresoccupying approximately one- to two-thirds of the area (26,37). High-resolution electron microscopic studies have pro-vided some knowledge of the dimensions and morphology ofthe pores running through the S layers. Information on themass distribution is in most cases limited to two-dimensionalprojections; recently, three-dimensional reconstructionshave been reported for a few S-layer types. Channels 2 to 3nm in diameter have been described for S layers of a varietyof mesophilic eubacteria (1, 3, 4, 8, 9, 11, 16, 32, 34-36). Bycontrast, larger channels with a limiting diameter of 5 to 6 nmhave been identified in S layers of some hyperthermophilicarchaebacteria (7, 19, 37). The structural information nowavailable clearly demonstrates that even among strains of thesame species the molecular shapes of the proteins making upthe S-layer lattices exhibit considerable differences (14, 18,31). Great diversity was observed within the protein domainsas well as within the fine, domain-linking protein bridges,which may serve to limit the size of the channels while stillenabling a high porosity.

Considering the data available on the structure, chemistry,and assembly of S layers, relatively little is known abouttheir functional significance. Due to their surface location, aprincipal role may be that of a promoter for cell adhesion andsurface recognition. S layers have also been shown tofunction as protective coats and molecular sieves (for re-views, see references 2, 27, and 28).

In the present study, the permeability properties and theexclusion limits of the S layers from two selected strains ofBacillus stearothermophilus were compared. Strains NRS1536/3c and PV72 were selected because their S layersexhibit remarkable differences with respect to their chemicaland morphological properties (18, 31).

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains and growth conditions. B. stearothermo-philus NRS 1536/3c and PV72 were grown in continuousculture as described previously (31).

Preparation of native and glutaraldehyde-treated sacculi.Whole cells (10 g, wet pellet) were suspended in 50 ml of aTriton X-100 solution (0.5% in 50 mM Tris hydrochloridebuffer, pH 7.2) and stirred at 300 rpm for 60 min at 20°C.After centrifugation at 10,000 x g, the pellet was suspendedin 30 ml of the Triton X-100 solution, extracted for 5 min at20°C, and centrifuged. The pellet obtained was washedthoroughly with distilled water and suspended, DNase andRNase (25 ,ug/ml) were added, and the suspension wasincubated for 30 min at 37°C. After centrifugation at 10,000x g, the pellet was washed with distilled water at least fivetimes. The material obtained by this procedure is referred toas native sacculi; these were used either for the permeabilitystudies or for the preparation of glutaraldehyde-treatedsacculi.For this purpose the pellet of native sacculi (5 g) was

suspended in 0.1 M sodium cacodylate buffer (pH 7.2), andthe final volume was adjusted to 49.0 ml. While the suspen-sion was stirred at 500 rpm at 20°C, 25% glutaraldehyde (1.0ml) was added. After 20 min the reaction was terminated bythe addition of 0.3 g of ethanolamine. Subsequently, thesuspension was centrifuged at 10,000 x g and repeatedlywashed with distilled water. Lysozyme (Boehringer Mann-heim Biochemicals; no. 107 255) was added (50 ,ug/ml), andthe mixture was incubated at 37°C for 60 min. The pellet wassedimented at 10,000 x g and repeatedly washed withdistilled water. The material obtained by this procedure isreferred to as glutaraldehyde-treated sacculi.

Native and glutaraldehyde-treated sacculi (1 g, wet pellet)were suspended in 8 ml of 5 mM Tris hydrochloride buffer(pH 7.0) and centrifuged at 40,000 x g for 20 min. After thisstep was repeated, the centrifuge tube was carefully wiped,and the pellet was weighed (+0.001 g).

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TABLE 1. Permeability of native and glutaraldehyde-treated B. stearothermophilus sacculi to probing moleculesof increasing molecular weight and sizea

Strain NRS 1536/3c sacculi Strain PV72 sacculi

SoluteMol Wtb PI Native ~~~~Glutaraldehyde NaieGlutaraldehydeSolute Mol wtb pI Native treated Native treated

Ss.1C Rsold Ss°} Rsol Ssol RSOl Ssol Rso

Mannose 180 76.3 68.9 83.2 78.2 88.7 83.8 89.8 86.4Myoglobin 17,900 6.8 73.3 65.0 75.1 67.6 80.3 74.2 79.5 72.4Carbonic anhydrase 30,000 5.3 70.0 60.7 72.1 63.7 80.3 71.7 71.9 62.2Ovalbumin 43,000 4.6 35.2 15.1 33.6 13.7 40.7 14.9 32.2 8.8Bovine serum albumin 67,000 4.7 26.8 4.0 24.2 1.4 33.4 4.4 28.2 3.5Dextran T-10 9,400 71.2 62.3 71.2 62.5 84.2 77.3 79.2 72.0Dextran T-40 42,000 33.1 12.3 33.7 13.8 46.4 23.0 36.3 14.4Dextran T-70 75,200 25.1 1.8 24.7 2.1 33.4 4.4 28.9 4.4Dextran T-510 450,000 23.7 23.1 30.3 25.6

a The values given represent the averages from six independent determinations. The highest standard deviation for the Ss,, values was ±4.5 for proteindetermination and ±3.6 for mannose or dextran determination.

b For the polydisperse dextran samples the weight-average molecular weight (M,,a) according to the manufacturer is given: Ma, = (N M,2/NjMj), where N, is thenumber of moles of the polymer species i, and Mi is the molecular weight of the species i.

' Ssol, Percentage total uptake values.d R,,,, Percentage corrected uptake values.

Negative staining, thin sectioning, and electron micros-copy were performed as described previously (18).

Labeling of native and glutaraldehyde-treated sacculi withferritin and polycationic ferritin. Labeling with ferritin andpolycationic ferritin (6) and freeze etching (18) were per-formed as described previously.

Permeability studies on native or glutaraldehyde-treatedsacculi. The permeability properties of native or glutaralde-hyde-treated sacculi were determined by the space tech-nique (24, 25).Packed pellets of native or glutaraldehyde-treated sacculi

were mixed with a known amount of solute (1 ml of 3%[wt/vol] concentration), and the mixture was allowed toequilibrate for 1 h at 20°C. The pellet was packed at 40,000 xg for 20 min, and the concentrations of the solutes in thesupernatants were determined.The solutes used for the space technique are listed in

Table 1. Proteins (Sigma, Munich, Federal Republic ofGermany) were determined by the method of Lowry et al.(17). Mannose (Sigma) and dextrans (Pharmacia, Uppsala,Sweden) were assayed by the orcine-sulfuric acid test (10).The percentage total uptake values for the selected solutes

(Ssol) and the percentage corrected uptake values (R501) werecalculated as given previously (24). Ss,, includes the totaluptake of a penetrating solute into the intracellular phase(interior of the sacculi) and the intercellular aqueous phaseof the packed pellet. To determine the intracellular phase(Sin) the high-molecular-weight dextran T-510 was used,because even the smallest molecules in this polydispersepreparation are too large to pass through the pores of theS-layer lattice. By substraction of the contents of the intra-cellular phase (Sin), the uptake of a given solute into theinterior of the sacculi can be calculated. This is expressed bythe corrected uptake value Rs.. The following equationswere used:

Ssol = (VsIWp)(CO1Cf- 1) X 100 (1)

Rsol = (Ssoi- Sin)(100- Sin) (2)

V, represents the volume of the solution added to the pellet,Wp is the weight of the pellet. C0 is the initial soluteconcentration and Cf is the final solute concentration as

measured in the supernatant after equilibration and repack-ing of the pellet.

Solute adsorption and solute degradation. To investigatesolute degradation, native sacculi (0.1 g) were suspended in1.0 ml of 5 mM Tris hydrochloride (pH 7.0), and 2 mg ofeither myoglobin, ovalbumin, or bovine serum albumin wasadded. The mixture was incubated for 1 h at 20°C andanalyzed by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis as previously described (18). To determine sol-ute adsorption, the firm pellet was suspended in a solutioncontaining 0.1, 0.5, or 1.0% (wt/vol) myoglobin or dextranT-10. The uptake values were calculated as described above.

RESULTS

Electron microscopy of whole cells. Electron micrographsof thin sections of whole cells of both B. stearothermophilusstrains exhibited a three-layered envelope structure (Fig. 1),composed of the cytoplasmic membrane, the peptidoglycan-containing layer, and the S layer as the outermost cell wallcomponent.

In freeze-etched preparations whole cells of both strainsappeared completely covered with crystalline arrays (Fig. 2).The S-layer lattice of strain NRS 1536/3c and that of strainPV72 displayed square (p4) and hexagonal (p6) symmetry,respectively (Fig. 2). The center-to-center spacing of themorphological units as derived from freeze-etched prepara-tions was 13.8 nm in strain NRS 1536/3c and 22.5 nm instrain PV72.

Electron microscopy of native and glutaraldehyde-treatedsacculi. In negatively stained preparations the Triton X-100-treated cells appeared as rod-shaped sacculi (data notshown). Thin-section preparations showed that all but tracesof the cytoplasmic membrane had been removed withoutnoticeably affecting the structural integrity of the peptido-glycan-containing layer and the S layer (Fig. 3a). Theseresults indicated that a disintegration of the cytoplasmicmembrane by mild detergent treatment did not induce cellwall autolysis in cells that were harvested in the logarithmicgrowth phase. In all preparations from which the cytoplas-mic membrane had been extracted, large fragments of addi-tional S-layer material were found to be attached to the innerface of the peptidoglycan-containing layer (Fig. 3a and b).

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The inner S layer was obviously formed from a surplus ofsubunits which came in contact with the peptidoglycan-containing layer after disintegration of the cytoplasmic mem-brane. Thus, native sacculi showed a three-layered envelopestructure composed of the outer S layer, the peptidoglycan-containing layer, and the incomplete, inner S layer (Fig. 3 aand b). As investigated with thin-section preparations, mostof the diffuse, granular material usually observed in theinterior of whole cells could be removed by nuclease treat-ment and extensive washing procedures (Fig. 3b). Digestionof the shape-determining peptidoglycan-containing layerwith lysozyme caused a disintegration of the native sacculi,accompanied by the release of S-layer fragments of a size ofup to 0.5 ,um. The S-layer lattice could be stabilized byglutaraldehyde treatment of native sacculi. Subsequent di-gestion of the peptidoglycan with lysozyme did not destroythe original shape of the sacculi (Fig. 3c). The glutaralde-hyde-treated sacculi exhibited a two-layered envelope struc-ture composed of the outer S layer and the inner S layer (Fig.3c). The distance between them resembled approximatelythe thickness of the original peptidoglycan-containing layer.

Labeling experiments. As determined by freeze-etching,the S-layer lattice on native sacculi of both strains did notbind polycationic ferritin (data not shown). By contrast, theS-layer surface of glutaraldehyde-treated sacculi was com-pletely covered with polycationic ferritin molecules (Fig. 4).The net negative charge required for binding of polycationicferritin was obviously generated by blocking the free aminogroups through reaction with glutaraldehyde. Neither nativenor glutaraldehyde-treated sacculi were capable of bindingthe negatively charged ferritin molecules. This indicated thatthe adsorption of negatively charged protein molecules tothe native or glutaraldehyde-treated S-layer surface wouldgenerally be low.

Electrophoretic evaluation. On sodium dodecyl sulfate-gelsthe S-layer proteins from B. stearothermophilus NRS1536/3c and PV72 gave strong bands with apparent molecu-lar weights of 117,000 (Fig. 5a) and 130,000 (Fig. Sc),respectively. Analysis of native sacculi of both organismsrevealed the same molecular weight of the S-layer proteinand lesser amounts of plasma proteins (Fig. 5 b and d). Afterincubation of the native sacculi with the proteins used for thepermeability studies two strong bands were detected onsodium dodecyl sulfate-gels, corresponding to the S-layerprotein and the respective, added protein (Fig. Se through g).Thus, any degradation of the added proteins during thenormal time scale of the permeability studies can be ex-cluded.

Solute uptake measurements. The intercellular space of thepacked pellet of native or glutaraldehyde-treated sacculi wasin the range of 30%, as determined with dextran T-510.Although the sacculi resembled rod-shaped bacterial cells,the intercellular space corresponded approximately to thetheoretical interspace of 27% for maximally packed,nondeformed spheres (5). The Sin values also demonstratedthat the sacculi were not markedly damaged during thepreparation procedure. This was in accordance with theelectron microscopic observations on thin-sectioned (Fig. 3)and negatively stained preparations.The maximal intracellular space into which a solute can

penetrate was determined with mannose, since the pores inthe S-layer lattice and those in the peptidoglycan-containingmeshwork must be large enough to allow an unhindereddiffusion for this small molecule. The percentage correcteduptake values (Rs.,) for mannose ranged from 68.9 to 86.4%(Table 1). In comparison with the mannose uptake values,

'-. .:-. .

FIG. 1. Thin section of whole cells of B. stearothermophilusNRS 1536/3c. cm, Cytoplasmic membrane; pg, peptidoglycan; s, Slayer. Bar, 200 nm.

FIG. 2. Freeze-etched preparations of intact cells of B. stearo-thermophilus strains (a) NRS 1536/3c and (b) PV72. Bars, 200 nm.

the uptake values for dextran T-10, myoglobin, and carbonicanhydrase were only slightly lower (Table 1). On the otherhand, the total uptake values (S,01) for dextran T-70 andbovine serum albumin were in the range of the values fordextran T-510 (Table 1). This indicated that these higher-molecular-weight solutes could not pass the permeabilitybarrier.By plotting the corrected uptake values (R,01) for the

monodisperse solutes, namely, mannose, myoglobin, car-bonic anhydrase, ovalbumin, or bovine serum albumin,versus the log of their molecular weights, a curve with a

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FIG. 4. Freeze-etched preparation of glutaraldehyde-treated sac-culi, labeled with polycationic ferritin. Bar, 200 nm.

Os is.N Wh . s .Sl_*_ ~~~~~~~~~~~~~~~~~~~~~~~~~.-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~kr,

Nf' hN.A

11

FIG. 3. Thin-sections of (a) Triton X-100-treated cells of B.stearothermophilus PV72 (b) native sacculi of strain PV72, and (c)glutaraldehyde-treated sacculi of strain NRS 1536/3c. pg, Peptido-glycan-containing layer; os, outer S layer; is, inner S layer. Bars,500 nm (a) and 200 nm (b and c).

sharp exclusion limit between molecular weights of 30,000and 45,000 was obtained (Fig. 6).As listed in Table 1, native and glutaraldehyde-treated

sacculi showed comparable permeability properties, indicat-ing that the exclusion limit was determined by the S-layerlattice and not by the peptidoglycan meshwork. Further-more, the permeability properties of the S layers of the twoB. stearothermophilus strains were rather similar (Table 1).The exclusion limits observed by using monodisperse

solutes were comparable to the exclusion limits obtainedwith the polydisperse dextran preparations. Due to theirmolecular-weight distribution and the fact the conformationsof the permeating dextrans are not known with certainty(13), it is difficult to correlate these findings. The R,01 valuesfor dextran T-40 may reflect the uptake of only the smallest

molecules present in the polydisperse preparation, whereasthe higher-molecular weight molecules may have been com-pletely excluded. In the case of dextran T-10 the sacculiwere evidently permeable for the whole range of moleculespresent in the preparation.When solute adsorption was studied, the R,01 values for

myoglobin were found to remain roughly constant at soluteconcentrations higher than 0.5% (wt/vol) (Table 2). At aconcentration of 0.1%, myoglobin was indeed adsorbed, butwith the solute concentrations employed for the permeabilitystudies adsorption should be completely obscured by theprevailing diffusional entry.

DISCUSSIONS layers have the potential to fulfill a broad spectrum of

functions (2, 27, 28). Due to their surface location and thefact that they completely cover the cell surface, they can actas protective coats and as barriers against movement ofexternal and internal macromolecules or as promoters forcell adhesion and surface recognition. The mass distributionof S layers of many species of eubacteria and archaebacteria

67 -

43

~~ b ~c d e gFIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electropho-

resis of extracts of (a) whole cells and (b) native sacculi of B.stearothermophilus NRS 1536/3c (c) whole cells and (d) nativesacculi of strain PV 72; or native sacculi of strain PV72 incubatedwith either (e) myoglobin (molecular weight, 17,000), (f) ovalbumin(43,000), or (g) bovine serum albumin (67,000).

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log MW

FIG. 6. Percentage uptake of solutes (R,0i) as a function of molecular weight by native sacculi (0) and glutaraldehyde-treated sacculi (A)prepared from whole cells of B. stearothermophilus strains (a) NRS 1536/3c and (b) PV72. Ma, Mannose; My, myoglobin; C, carbonicanhydrase, 0, ovalbumin; BSA, bovine serum albumin.

has been studied by high-resolution electron microscopyincluding image processing procedures (1, 3, 4, 7-9, 11, 16,19, 26, 32, 34,-37). Although resolutions below 2 nm can beattained by these methods, the information obtained doesnot suffice to accurately predict the molecular sieving prop-erties and the exclusion limits of these S layers. The molec-ular sieving by crystalline arrays of protein or glycoproteinsubunits will imply interactions of the solute itself with theprotein matrix as well as with the carbohydrate moietycovalently attached to the polypeptide chain (15). In addi-tion, the protein domains of the pore area may be assumed toundergo reversible conformational changes under certainenvironmental conditions. From such conformationalchanges, alterations of the pore morphology and pore sizewould ensue which would not likely be detected by thecommonly applied electron microscopic examination proce-dures. Indeed, preliminary studies on isolated S layers ofdifferent members of the family Bacillaceae indicated thattheir molecular sieving properties are influenced by the pHand the ionic strength of the environment (30). Therefore,the permeability studies with structurally well-defined sol-utes appear as a promising approach to the determination ofthe effective permeability properties and exclusion limits ofS layers under well-defined environmental conditions.

If S layers function as protective coats and as molecularsieves, a different kind of selection pressure would haveoptimized these protein layers during evolution. For variousspecies of mesophilic eubacteria, it was suggested that thepores running through their S layers are smaller than 3 nm.

TABLE 2. Percentage corrected uptake (R,01) for myoglobin anddextran T-10 by glutaraldehyde-treated sacculi of

B. stearothermophilus NRS 1536/3c, as afunction of the solute concentration

RsoSolute concn (%)

Myoglobin Dextran T-10

0.1 Adsorption 80.00.5 83.2 79.61.0 76.2 75.83.0 75.6 77.6

In these cases, the S layers could function as a protectivecoat against lysogenic enzymes such as lysozyme and pro-teases (2, 21, 28, 38). As recently observed, such a protec-tive function against lysozyme attack can be ruled out atleast for S layers of thermophilic members of the Bacil-laceae, such as B. stearothermophilus (18), Desutfotoma-culum nigrificsans (31), Clostridium thermohydrosulfuricum(30), and Clostridium thermosaccharolyticum (unpublishedobservations). All strains examined so far were highly sen-sitive to lysozyme, indicating that these S layers do notreject molecules with a size of up to 3.5 nm (18, 30).Interestingly, S layers on intact cells showed a remarkabledegree of resistance against proteolytic digestion, whichmight be at least in part explained by the presence of glycanchains on the promoters (28, 31).For both the hexagonal and square S-layer lattice investi-

gated in this study, exclusion limits between molecularweights of 30,000 and 45,000 were observed. Obviously,similar pores can be generated by the self-assembly ofmorphologically different protomers. Therefore, the diver-sity among S layers of the same species with respect to theirchemical and morphological properties is not viewed as inconflict with an identity of functional significance such asthat of a molecular sieve. If the carbonic anhydrase molecule(molecular weight 30,000) with a dimension of 4.1 by 4.1 by4.7 nm (33) penetrates in the lengthwise orientation, the poresizes in the S layers of both B. stearothermophilus strainsmust exceed 4.1 nm. Pore sizes in the range of even 5 to 6 nmhave been determined for the S layers of a few hyperther-mophilic archaebacteria (7, 19, 37). Due to the fact that thesearchaebacteria and the highly thermophilic members of theBacillaceae grow under very selective environmental condi-tions, selection pressures other than the presence of lyticenzymes must have determined the pore sizes of their Slayers.Most strains of B. stearothermophilus, including the two

strains examined in this study, secrete large amounts ofexoenzymes such as amylases, lipases, and proteases (20)generally ranging in molecular weight from 20,000 to 40,000(12). Thus, the S-layer pores of strains NRS 1536/3c andPV72 would allow free passage for most exoproteins, whichare presumed to undergo their final folding during passagethrough the peptidoglycan layer (22). Of relevance in this

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context is the observation that the peptidoglycan-containinglayer does not limit the passage of molecules capable ofpenetrating the S layer.The information now available from structural and perme-

ability studies on S layers from both B. stearothermophilusstrains and other selected thermophilic members of theBacillaceae makes it unlikely that their S layers have thepotential to function as an effective barrier against mostlysogenic enzymes. On the other hand there must be a strongselection pressure for maintaining S layers in competitiveenvironments, since during continuous growth under opti-mal laboratory conditions wild-type strains are frequentlyoutgrown by S layer-deficient mutants (28).

S layers of all B. stearothermophilus strains studied so farwere found to differ in their surface net charge from that ofthe peptidoglycan-containing sacculus (23). By masking thenet negatively charged, rigid cell wall layer, the S layer willmediate surface recognition and surface adsorption phenom-ena and the interactions between the living cell and thenutrients present in the medium. The permeability studies onS layers of highly thermophilic members of the Bacillaceaeclearly demonstrate that the pore areas in the protein mesh-work have a low tendency for unspecific adsorption ofmacromolecules (30). This is essential for maintaining anunhindered exchange of nutrients and mnetabolites betweenthe cell and its environment.

Aside from these considerations the specific biologicalsignificance of S layers of the highly thermophilic membersof the Bacillaceae is yet unknown. The remarkable diversityobserved among strains of even the same species demon-strates that many variants of these two-dimensional, crys-talline arrays have arisen in context with similar survivalstrategies during evolution. Further studies will showwhether, apart from these diversities, structural and func-tional elements have been conserved in those domains of theS-layer protomers that correspond to the pore area.

ACKNOWLEDGMENTS

We thank F. M. Unger for critical reading of the manuscript.This work was supported by Osterreichischer Fonds zur

Forderung der wissenschaftlichen Forschung, project 5290 and bythe Bundesministerium fur Wissenschaft und Forschung.

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