1887

Abstract

The mature surface layer (S-layer) protein SbsC of ATCC 12980 comprises amino acids 31–1099 and self-assembles into an oblique lattice type which functions as an adhesion site for a cell-associated high-molecular-mass exoamylase. To elucidate the structure–function relationship of distinct segments of SbsC, three N- and seven C-terminal truncations were produced in a heterologous expression system, isolated, purified and their properties compared with those of the recombinant mature S-layer protein rSbsC. With the various truncated forms it could be demonstrated that the N-terminal part (aa 31–257) is responsible for anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, but this positively charged segment is not required for the self-assembly of SbsC, nor for generating the oblique lattice structure. If present, the N-terminal part leads to the formation of double-layer self-assembly products. Affinity studies further showed that the N-terminal part includes an exoamylase-binding site. Interestingly, the N-terminal part carries two sequences of 6 and 7 aa (AKAALD and KAAYEAA) that were also identified on the amylase-binding protein AbpA of . In contrast to the self-assembling N-terminal truncation rSbsC, two further N-terminal truncations (rSbsC, rSbsC) and three C-terminal truncations (rSbsC, rSbsC, rSbsC) had lost the ability to self-assemble and stayed in the water-soluble state. Studies with the self-assembling C-terminal truncations rSbsC, rSbsC and rSbsC revealed that the C-terminal 219 aa can be deleted without interfering with the self-assembly process, while the C-terminal 179 aa are not required for the formation of the oblique lattice structure.

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2001-05-01
2024-05-03
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References

  1. Archibald A. R., Hancock I. C., Harwood C. R. 1993; Cell wall structure, synthesis, and turnover. In Bacillus subtilis and Other Gram-Positive Bacteria pp 381–410 Edited by Sonenshein A., Hoch J. A., Losick R. New York: Academic Press;
    [Google Scholar]
  2. Brechtel E., Bahl H. 1999; In Thermoanaerobacterium thermosulfurigenes EM1 S-layer homology domains do not attach to peptidoglycan. J Bacteriol 181:5017–5023
    [Google Scholar]
  3. Chami M., Bayan N., Peyret J. L., Gulik-Krzywicki T., Leblon G., Shechter E. 1997; The S-layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C-terminal hydrophobic domain. Mol Microbiol 23:483–492 [CrossRef]
    [Google Scholar]
  4. Chauvaux S., Matuschek M., Béguin P. 1999; Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis . J Bacteriol 181:2455–2458
    [Google Scholar]
  5. Egelseer E. M., Schocher I., Sleytr U. B., Sára M. 1996; Evidence that an N-terminal S-layer protein fragment triggers the release of a cell wall-associated high-molecular weight amylase from Bacillus stearothermophilus ATCC 12980. J Bacteriol 178:5602–5609
    [Google Scholar]
  6. Egelseer E. M., Schocher I., Sleytr U. B., Sára M. 1997; Evidence that the S-layer of Bacillus stearothermophilus strains functions as an adhesion site for a high-molecular mass amylase. FEMS Microbiol Rev 20:114–118
    [Google Scholar]
  7. Egelseer E. M., Leitner K., Jarosch M., Hotzy C., Zayni S., Sleytr U. B., Sára M. 1998; The S-layer proteins of two Bacillus stearothermophilus wild-type strains are bound via their N-terminal region to a secondary cell wall polymer of identical chemical composition. J Bacteriol 180:1488–1495
    [Google Scholar]
  8. Etienne-Toumelin I., Sirard J.-C., Duflot E., Mock M., Fouet A. 1995; Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene. J Bacteriol 177:614–620
    [Google Scholar]
  9. Ilk N., Kosma P., Puchberger M., Egelseer E. M., Mayer H. F., Sleytr U. B., Sára M. 1999; Complete structural analysis and biological function of the secondary cell wall polymer of Bacillus sphaericus CCM 2177 serving as an S-layer specific anchor. J Bacteriol 181:7643–7646
    [Google Scholar]
  10. Jarosch M., Egelseer E. M., Mattanovich D., Sleytr U. B., Sára M. 2000; S-layer gene sbsC of Bacillus stearothermophilus ATCC 12980: molecular characterization and heterologous expression in Escherichia coli . Microbiology 146:273–281
    [Google Scholar]
  11. Lacks S. A., Springhorn S. S. 1980; Renaturation of enyzmes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J Biol Chem 255:7467–7473
    [Google Scholar]
  12. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  13. Lemaire M., Miras I., Gounon P., Béguin P. 1998; Identification of a region responsible for binding to the cell wall within the S-layer protein of Clostridium thermocellum . Microbiology 144:211–217 [CrossRef]
    [Google Scholar]
  14. Mesnage S., Tosi-Couture E., Fouet A. 1999; Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S-layer proteins and the Bacillus subtilis levansucrase. Mol Microbiol 31:927–936 [CrossRef]
    [Google Scholar]
  15. Messner P., Hollaus F., Sleytr U. B. 1984; Paracrystalline cell wall surface layers of different Bacillus stearothermophilus strains. Int J Syst Bacteriol 34:202–210 [CrossRef]
    [Google Scholar]
  16. Messner P., Pum D., Sleytr U. B. 1986; Characterization of the ultrastructure and the self-assembly of the surface layer of Bacillus stearothermophilus strain NRS 2004/3a. J Ultrastuct Mol Struct Res 97:73–88 [CrossRef]
    [Google Scholar]
  17. Olabarrı́a G., Carrascosa J. L., De Pedro M. A., Berenguer J. 1996; A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus. J Bacteriol 178:4765–4772
    [Google Scholar]
  18. Ries W., Hotzy C., Schocher I., Sleytr U. B., Sára M. 1997; Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer. J Bacteriol 179:3892–3898
    [Google Scholar]
  19. Rogers J. D., Haase E. M., Brown A. E., Douglas C. W. I., Gwynn J. P., Scannapieco F. A. 1998; Identification and analysis of a gene ( abpA ) encoding a major amylase-binding protein in Streptococcus gordonii . Microbiology 144:1223–1233 [CrossRef]
    [Google Scholar]
  20. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Sára M. Sleytr U. B. 2000; S-layer proteins. J Bacteriol 182:859–868 [CrossRef]
    [Google Scholar]
  22. Sára M. Kuen B., Mayer H. F., Mandl F., Schuster K. C., Sleytr U. B. 1996; Dynamics in oxygen-induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and its S-layer deficient variant T5 in continuous culture and studies on the cell wall composition. J Bacteriol 178:2108–2117
    [Google Scholar]
  23. Sára M. Dekitsch C., Mayer H. F., Egelseer E. M., Sleytr U. B. 1998; Influence of the secondary cell wall polymer on the reassembly, recrystallization and stability properties of the S-layer protein from Bacillus stearothermophilus PV72/p2. J Bacteriol 180:4146–4153
    [Google Scholar]
  24. Schäffer C., Kählig H. Christian R., Schulz G., Zayni S., Messner P. 1999; The diacetamidodideoxyuronic acid-containing glycan chain of Bacillus stearothermophilus NRS 2004/3a represents the secondary cell wall polymer of wild-type B. stearothermophilus strains. Microbiology 145:1575–1583 [CrossRef]
    [Google Scholar]
  25. Sleytr U. B., Beveridge T. J. 1999; Bacterial S-layers. Trends Microbiol 7:253–260 [CrossRef]
    [Google Scholar]
  26. Sleytr U. B., Messner P., Pum D., Sára M. 1999; Crystalline bacterial cell surface layers (S-layers): from cell structure to biomimetics and nanotechnology. Angew Chem Int Ed Engl 38:1034–1054 [CrossRef]
    [Google Scholar]
  27. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
    [Google Scholar]
  28. Thomas S., Austin J. W., McCubbin W. D., Kay C. M., Trust T.J. 1992; Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. J Mol Biol 228:652–661 [CrossRef]
    [Google Scholar]
  29. Transue T. R., Smith K. A., Mo H., Goldstein I. J., Saper M. A. 1997; Structure of benzyl-T-antigen disaccharide bound to Amaranthus caudatus agglutinin. Nature Struct Biol 4:779–783 [CrossRef]
    [Google Scholar]
  30. Trust T. J. 1993; Molecular, structural and functional properties of Aeromonas S-layers. In Advances in Bacterial Paracrystalline Surface Layers pp 159–173 Edited by Beveridge T. J., Koval S. F. New York: Plenum;
    [Google Scholar]
  31. Weis W. I. 1997; Cell-surface carbohydrate recognition by animal and viral lectins. Curr Opin Struct Biol 7:624–630 [CrossRef]
    [Google Scholar]
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