1887

Abstract

, the aetiological agent of anthrax, synthesizes two surface-layer (S-layer) proteins. S-layers are two-dimensional crystalline arrays that completely cover bacteria. In rich medium, the S-layer consists of Sap during the exponential growth phase. Sap is a modular protein composed of an SLH (S-layer homology)-anchoring domain followed by a putative crystallization domain (Sap). A projection map of the two-dimensional Sap array has been established on deflated bacteria. In this work, the authors used two approaches to investigate whether Sap is the crystallization domain. The purified Sap polypeptide (604 aa) was sufficient to form a crystalline structure, as illustrated by electron microscopy. Consistent with this result, the entire Sap domain promoted auto-interaction in a bacterial two-hybrid screen developed for the present study. The screen was derived from a system that takes advantage of the cyclase subdomain structure to enable one to identify peptides that interact. A screening strategy was then employed to study Sap subdomains that mediate interaction. A random library, derived from the Sap domain, was constructed and screened. The selected polypeptides interacting with the complete Sap were all larger (155 aa and above) than the mean size of the randomly cloned peptides (approx. 60 residues). This result suggests that, in contrast with observations for other interactions studied with this two-hybrid system, large fragments were required to ensure efficient interaction. It was noteworthy that only one polypeptide, which spanned aa 148–358, was able to interact with less than the complete Sap, in fact, with itself.

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2005-05-01
2024-03-28
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References

  1. Baumeister W., Wildhaber I., Phipps B. M. 1989; Principles of organization in eubacterial and archaebacterial surface proteins. Can J Microbiol 35:215–227 [CrossRef]
    [Google Scholar]
  2. Bingle W. H., Engelhardt H., Page W. J., Baumeister W. 1987; Three-dimensional structure of the regular tetragonal surface layer of Azotobacter vinelandii . J Bacteriol 169:5008–5015
    [Google Scholar]
  3. Chung C. T., Miller R. H. 1988; A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res 16:3580 [CrossRef]
    [Google Scholar]
  4. Claus H., Akca E., Debaerdemaeker T., Evrard C., Declercq J. P., Konig H. 2002; Primary structure of selected archaeal mesophilic and extremely thermophilic outer surface layer proteins. Syst Appl Microbiol 25:3–12 [CrossRef]
    [Google Scholar]
  5. Couture-Tosi E., Delacroix H., Mignot T., Mesnage S., Chami M., Fouet A., Mosser G. 2002; Structural analysis and evidence for dynamic emergence of Bacillus anthracis S-layer networks. J Bacteriol 184:6448–6456 [CrossRef]
    [Google Scholar]
  6. 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]
  7. Engelhardt H., Peters J. 1998; Structural research on surface layers: a focus on stability, surface layer homology domains, and surface layer cell wall interactions. J Struct Biol 124:276–302 [CrossRef]
    [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. Gilmour M. W., Gunton J. E., Lawley T. D., Taylor D. E. 2003; Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB. Mol Microbiol 49:105–116 [CrossRef]
    [Google Scholar]
  10. Gropp M., Strausz Y., Gross M., Glaser G. 2001; Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain. J Bacteriol 183:570–579 [CrossRef]
    [Google Scholar]
  11. Howorka S., Wang Y. J., Kuen B., Sleytr U. B., Lubitz W., Bayley H, Sára M. 2000; Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein. J Biol Chem 275:37876–37886 [CrossRef]
    [Google Scholar]
  12. Ilk N., Kosma P., Puchberger M., Egelseer E. M., Mayer H. F., Sleytr U. B., Sára M. 1999; Structural and functional analyses of the secondary cell wall polymer of Bacillus sphaericus CCM 2177 that serves as an S-layer-specific anchor. J Bacteriol 181:7643–7646
    [Google Scholar]
  13. 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 inEscherichia coli . Microbiology 146:273–281
    [Google Scholar]
  14. Jarosch M., Egelseer E. M., Huber C., Moll D., Mattanovich D., Sleytr U. B., Sára M. 2001; Analysis of the structure–function relationship of the S-layer protein SbsC of Bacillus stearothermophilus ATCC 12980 by producing truncated forms. Microbiology 147:1353–1363
    [Google Scholar]
  15. Jing H., Takagi J., Liu J. H., Lindgren S., Zhang R. G., Joachimiak A., Wang J. H., Springer T. A. 2002; Archaeal surface layer proteins contain β propeller, PKD, and β helix domains and are related to metazoan cell surface proteins. Structure 10:1453–1464 [CrossRef]
    [Google Scholar]
  16. Jobling M. G., Holmes R. K. 2000; Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system. Proc Natl Acad Sci U S A 97:14662–14667 [CrossRef]
    [Google Scholar]
  17. Karimova G., Pidoux J., Ullmann A., Ladant D. 1998; A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95:5752–5756 [CrossRef]
    [Google Scholar]
  18. Karimova G., Ullmann A., Ladant D. 2000; Bordetella pertussis adenylate cyclase toxin as a tool to analyze molecular interactions in a bacterial two-hybrid system. Int J Med Microbiol 290:441–445 [CrossRef]
    [Google Scholar]
  19. Karimova G., Ullmann A., Ladant D. 2001; Protein–protein interaction between Bacillus stearothermophilus tyrosyl-tRNA synthetase subdomains revealed by a bacterial two-hybrid system. J Mol Microbiol Biotechnol 3:73–82
    [Google Scholar]
  20. Lupas A., Engelhardt H., Peters J., Santarius U., Volker S., Baumeister W. 1994; Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis. J Bacteriol 176:1224–1233
    [Google Scholar]
  21. Mader C., Huber C., Moll D., Sleytr U. B., Sára M. 2004; Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology. J Bacteriol 186:1758–1768 [CrossRef]
    [Google Scholar]
  22. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Mesnage S., Tosi-Couture E., Mock M., Gounon P., Fouet A. 1997; Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen. Mol Microbiol 23:1147–1155 [CrossRef]
    [Google Scholar]
  24. 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]
  25. Mesnage S., Fontaine T., Mignot T., Delepierre M., Mock M., Fouet A. 2000; Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation. EMBO J 19:4473–4484 [CrossRef]
    [Google Scholar]
  26. Mignot T., Mesnage S., Couture-Tosi E., Mock M., Fouet A. 2002; Developmental switch of S-layer protein synthesis in Bacillus anthracis. Mol Microbiol 43:1615–1627 [CrossRef]
    [Google Scholar]
  27. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  28. Pavkov T., Oberer M., Egelseer E. M., Sleytr U. B., Keller W, Sára M. 2003; Crystallization and preliminary structure determination of the C-terminal truncated domain of the S-layer protein SbsC. Acta Cryst D59:1466–1468
    [Google Scholar]
  29. Rünzler D., Huber C., Moll D., Köhler G., Sára M. 2004; Biophysical characterization of the entire bacterial surface layer protein SbsB and its two distinct functional domains. J Biol Chem 279:5207–5215 [CrossRef]
    [Google Scholar]
  30. Sára M., Sleytr U. B. 2000; S-layer proteins. J Bacteriol 182:859–868 [CrossRef]
    [Google Scholar]
  31. 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 the S-layer-deficient variant T5 in continuous culture and studies of the cell wall composition. J Bacteriol 178:2108–2117
    [Google Scholar]
  32. Sillanpää J., Martínez B., Antikainen J. 9 other authors 2000; Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus . J Bacteriol 182:6440–6450 [CrossRef]
    [Google Scholar]
  33. Smit E., Oling F., Demel R., Martinez B., Pouwels P. H. 2001; The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J Mol Biol 305:245–257 [CrossRef]
    [Google Scholar]
  34. Smit E., Jager D., Martinez B., Tielen F. J., Pouwels P. H. 2002; Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356: evidence for protein–protein interaction of two subdomains. J Mol Biol 324:953–964 [CrossRef]
    [Google Scholar]
  35. Smith J. M. 1999; Ximdisp – a visualization tool to aid structure determination from electron microscope images. J Struct Biol 125:223–228 [CrossRef]
    [Google Scholar]
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