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Volume 156, Issue 1

Processing, assembly and localization of a spore protein Free

Abstract

All spores are encased in macromolecular shells. One of these is a proteinacious shell called the coat that, in , provides critical protective functions. The spore is the infectious particle for the disease anthrax. Therefore, the coat is of particular interest because it may provide essential protective functions required for the appearance of anthrax. Here, we analyse a protein component of the spore outer layers that was previously designated BxpA. Our data indicate that a significant amount of BxpA is located below the spore coat and associated with the cortex. By SDS-PAGE, BxpA migrates as a 9 kDa species when extracted from Sterne strain spores, and as 11 and 14 kDa species from Ames strain spores, even though it has predicted masses of 27 and 29 kDa, respectively, in these two strains. We investigated the possibility that BxpA is subject to post-translational processing as previously suggested. In , a subset of coat proteins is proteolysed or cross-linked by the spore proteins YabG or Tgl, respectively. To investigate the possibility that similar processing occurs in , we generated mutations in the or genes in the Sterne and Ames strains and analysed the consequences for BxpA assembly by SDS-PAGE. We found that in a mutant of the apparent mass of BxpA increased. This is consistent with the possibility that Tgl directs the cross-linking of BxpA into a form that normally does not enter the gel. Unexpectedly, the apparent mass of BxpA also increased in a mutant, suggesting a relatively complex role for proteolysis in spore protein maturation in . These data reveal a previously unobserved event in spore protein maturation in . We speculate that proteolysis and cross-linking are ubiquitous spore assembly mechanisms throughout the genus .

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): IEM, immuno-electron microscopy and TEM, transmission electron microscopy
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2010-01-01
2024-04-25
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References

  1. Arantes O., Lereclus D. 1991; Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119
     [Google Scholar]
  2. Aronson A. I., Fitz-James P. C. 1968; Biosynthesis of bacterial spore coats. J Mol Biol 33:199–212
     [Google Scholar]
  3. Aronson A. I., Fitz-James P. 1976; Structure and morphogenesis of the bacterial spore coat. Bacteriol Rev 40:360–402
     [Google Scholar]
  4. Bergman N. H., Anderson E. C., Swenson E. E., Niemeyer M. M., Miyoshi A. D., Hanna P. C. 2006; Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation. J Bacteriol 188:6092–6100
     [Google Scholar]
  5. Boydston J. A., Yue L., Kearney J. F., Turnbough C. L. Jr 2006; The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis. J Bacteriol 188:7440–7448
     [Google Scholar]
  6. Bozue J. A., Parthasarathy N., Phillips L. R., Cote C. K., Fellows P. F., Mendelson I., Shafferman A., Friedlander A. M. 2005; Construction of a rhamnose mutation in Bacillus anthracis affects adherence to macrophages but not virulence in guinea pigs. Microb Pathog 38:1–12
     [Google Scholar]
  7. Bozue J., Cote C. K., Moody K. L., Welkos S. L. 2007a; Fully virulent Bacillus anthracis does not require the immunodominant protein BclA for pathogenesis. Infect Immun 75:508–511
     [Google Scholar]
  8. Bozue J., Moody K. L., Cote C. K., Stiles B. G., Friedlander A. M., Welkos S. L., Hale M. L. 2007b; Bacillus anthracis spores of the bclA mutant exhibit increased adherence to epithelial cells, fibroblasts, and endothelial cells but not to macrophages. Infect Immun 75:4498–4505
     [Google Scholar]
  9. Cote C. K., Rossi C. A., Kang A. S., Morrow P. R., Lee J. S., Welkos S. L. 2005; The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions. Microb Pathog 38:209–225
     [Google Scholar]
  10. Cote C. K., Bozue J., Moody K. L., DiMezzo T. L., Chapman C. E., Welkos S. L. 2008; Analysis of a novel spore antigen in Bacillus anthracis that contributes to spore opsonization. Microbiology 154:619–632
     [Google Scholar]
  11. Cutting S., Zheng L. B., Losick R. 1991; Gene encoding two alkali-soluble components of the spore coat from Bacillus subtilis. J Bacteriol 173:2915–2919
     [Google Scholar]
  12. Driks A. 1999; Bacillus subtilis spore coat. Microbiol Mol Biol Rev 63:1–20
     [Google Scholar]
  13. Driks A. 2002; Maximum shields: the assembly and function of the bacterial spore coat. Trends Microbiol 10:251–254
     [Google Scholar]
  14. Driks A. 2003; The dynamic spore. Proc Natl Acad Sci U S A 100:3007–3009
     [Google Scholar]
  15. Fellows P. F., Linscott M. K., Ivins B. E., Pitt M. L., Rossi C. A., Gibbs P. H., Friedlander A. M. 2001; Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19:3241–3247
     [Google Scholar]
  16. Friedlander A. M. 2000; Anthrax: clinical features, pathogenesis, and potential biological warfare threat. Curr Clin Top Infect Dis 20:335–349
     [Google Scholar]
  17. Gerhardt P. 1967; Cytology of Bacillus anthracis. Fed Proc 26:1504–1517
     [Google Scholar]
  18. Gerhardt P., Ribi E. 1964; Ultrastructure of the exosporium enveloping spores of Bacillus cereus. J Bacteriol 88:1774–1789
     [Google Scholar]
  19. Giorno R., Bozue J., Cote C., Wenzel T., Moody K. S., Mallozzi M., Ryan M., Wang R., Zielke R. other authors 2007; Morphogenesis of the Bacillus anthracis spore. J Bacteriol 189:691–705
     [Google Scholar]
  20. Goldman R. C., Tipper D. J. 1978; Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component. J Bacteriol 135:1091–1106
     [Google Scholar]
  21. Henriques A. O., Moran C. P. Jr 2000; Structure and assembly of the bacterial endospore coat. Methods 20:95–110
     [Google Scholar]
  22. Henriques A. O., Moran C. P. Jr 2007; Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol 61:555–588
     [Google Scholar]
  23. Hines H. B., Lebeda F., Hale M., Brueggemann E. E. 2005; Characterization of botulinum progenitor toxins by mass spectrometry. Appl Environ Microbiol 71:4478–4486
     [Google Scholar]
  24. Johnson M. J., Todd S. J., Ball D. A., Shepherd A. M., Sylvestre P., Moir A. 2006; ExsY and CotY are required for the correct assembly of the exosporium and spore coat of Bacillus cereus. J Bacteriol 188:7905–7913
     [Google Scholar]
  25. Kang T. J., Fenton M. J., Weiner M. A., Hibbs S., Basu S., Baillie L., Cross A. S. 2005; Murine macrophages kill the vegetative form of Bacillus anthracis. Infect Immun 73:7495–7501
     [Google Scholar]
  26. Keim P., Price L. B., Klevytska A. M., Smith K. L., Schupp J. M., Okinaka R., Jackson P. J., Hugh-Jones M. E. 2000; Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol 182:2928–2936
     [Google Scholar]
  27. Kobayashi K., Hashiguchi K., Yokozeki K., Yamanaka S. 1998; Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli. Biosci Biotechnol Biochem 62:1109–1114
     [Google Scholar]
  28. Kuwana R., Okuda N., Takamatsu H., Watabe K. 2006; Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores. J Biochem 139:887–901
     [Google Scholar]
  29. Kuwana R., Takamatsu H., Watabe K. 2007; Expression, localization and modification of YxeE spore coat protein in Bacillus subtilis. J Biochem 142:681–689
     [Google Scholar]
  30. Little S., Driks A. 2001; Functional analysis of the Bacillus subtilis morphogenetic spore coat protein CotE. Mol Microbiol 42:1107–1120
     [Google Scholar]
  31. Little S. F., Knudson G. B. 1986; Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun 52:509–512
     [Google Scholar]
  32. Liu H., Bergman N. H., Thomason B., Shallom S., Hazen A., Crossno J., Rasko D. A., Ravel J., Read T. D. other authors 2004; Formation and composition of the Bacillus anthracis endospore. J Bacteriol 186:164–178
     [Google Scholar]
  33. Lyons C. R., Lovchik J., Hutt J., Lipscomb M. F., Wang E., Heninger S., Berliba L., Garrison K. 2004; Murine model of pulmonary anthrax: kinetics of dissemination, histopathology, and mouse strain susceptibility. Infect Immun 72:4801–4809
     [Google Scholar]
  34. Mallozzi M., Bozue J., Giorno R., Moody K. S., Slack A., Cote C., Qiu D., Wang R., McKenney P. other authors 2008; Characterization of a Bacillus anthracis spore coat-surface protein that influences coat-surface morphology. FEMS Microbiol Lett 289:110–117
     [Google Scholar]
  35. Mendelson I., Tobery S., Scorpio A., Bozue J., Shafferman A., Friedlander A. M. 2004; The NheA component of the non-hemolytic enterotoxin of Bacillus cereus is produced by Bacillus anthracis but is not required for virulence. Microb Pathog 37:149–154
     [Google Scholar]
  36. Monroe A., Setlow P. 2006; Localization of the transglutaminase cross-linking sites in the Bacillus subtilis spore coat protein GerQ. J Bacteriol 188:7609–7616
     [Google Scholar]
  37. Ozin A. J., Henriques A. O., Yi H., Moran C. P. Jr 2000; Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat. J Bacteriol 182:1828–1833
     [Google Scholar]
  38. Pandey N. K., Aronson A. I. 1979; Properties of the Bacillus subtilis spore coat. J Bacteriol 137:1208–1218
     [Google Scholar]
  39. Perez-Casal J., Caparon M. G., Scott J. R. 1991; Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol 173:2617–2624
     [Google Scholar]
  40. Popham D. L. 2002; Specialized peptidoglycan of the bacterial endospore: the inner wall of the lockbox. Cell Mol Life Sci 59:426–433
     [Google Scholar]
  41. Ragkousi K., Eichenberger P., van Ooij C., Setlow P. 2003; Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate. J Bacteriol 185:2315–2329
     [Google Scholar]
  42. Read T. D., Peterson S. N., Tourasse N., Baillie L. W., Paulsen I. T., Nelson K. E., Tettelin H., Fouts D. E., Eisen J. A. other authors 2003; The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423:81–86
     [Google Scholar]
  43. Redmond C., Baillie L. W., Hibbs S., Moir A. J., Moir A. 2004; Identification of proteins in the exosporium of Bacillus anthracis. Microbiology 150:355–363
     [Google Scholar]
  44. Riesenman P. J., Nicholson W. L. 2000; Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation. Appl Environ Microbiol 66:620–626
     [Google Scholar]
  45. Schaeffer P., Millet J., Aubert J. P. 1965; Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54:704–711
     [Google Scholar]
  46. Setlow P. 2006; Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101:514–525
     [Google Scholar]
  47. Severson K. M., Mallozzi M., Bozue J., Welkos S. L., Cote C. K., Knight K. L., Driks A. 2009; Roles of the Bacillus anthracis spore protein ExsK in exosporium maturation and germination. J Bacteriol Oct 16: [Epub ahead of print]
    [Google Scholar]
  48. Steichen C., Chen P., Kearney J. F., Turnbough C. L. Jr 2003; Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J Bacteriol 185:1903–1910
     [Google Scholar]
  49. Steichen C. T., Kearney J. F., Turnbough C. L. Jr 2005; Characterization of the exosporium basal layer protein BxpB of Bacillus anthracis. J Bacteriol 187:5868–5876
     [Google Scholar]
  50. Sylvestre P., Couture-Tosi E., Mock M. 2003; Polymorphism in the collagen-like region of the Bacillus anthracis BclA protein leads to variation in exosporium filament length. J Bacteriol 185:1555–1563
     [Google Scholar]
  51. Sylvestre P., Couture-Tosi E., Mock M. 2005; Contribution of ExsFA and ExsFB proteins to the localization of BclA on the spore surface and to the stability of the Bacillus anthracis exosporium. J Bacteriol 187:5122–5128
     [Google Scholar]
  52. Takamatsu H., Kodama T., Imamura A., Asai K., Kobayashi K., Nakayama T., Ogasawara N., Watabe K. 2000; The Bacillus subtilis yabG gene is transcribed by SigK RNA polymerase during sporulation, and yabG mutant spores have altered coat protein composition. J Bacteriol 182:1883–1888
     [Google Scholar]
  53. Takamatsu H., Imamura D., Kuwana R., Watabe K. 2009; Expression of yeeK during Bacillus subtilis sporulation and localization of YeeK to the inner spore coat using fluorescence microscopy. J Bacteriol 191:1220–1229
     [Google Scholar]
  54. Thorne C. B. 1968; Transducing bacteriophage for Bacillus cereus. J Virol 2:657–662
     [Google Scholar]
  55. Todd S. J., Moir A. J., Johnson M. J., Moir A. 2003; Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. J Bacteriol 185:3373–3378
     [Google Scholar]
  56. Vary P. S. 1994; Prime time for Bacillus megaterium. Microbiology 140:1001–1013
     [Google Scholar]
  57. Welkos S. L., Cote C. K., Rea K. M., Gibbs P. H. 2004; A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies. J Microbiol Methods 56:253–265
     [Google Scholar]
  58. Williams D. D., Turnbough C. L. Jr 2004; Surface layer protein EA1 is not a component of Bacillus anthracis spores but is a persistent contaminant in spore preparations. J Bacteriol 186:566–569
     [Google Scholar]
  59. Zilhao R., Isticato R., Martins L. O., Steil L., Volker U., Ricca E., Moran C. P. Jr, Henriques A. O. 2005; Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis. J Bacteriol 187:7753–7764
     [Google Scholar]
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Processing, assembly and localization of a Bacillus anthracis spore protein
Microbiology 156, 174 (2010); https://doi.org/10.1099/mic.0.033407-0
/content/journal/micro/10.1099/mic.0.033407-0
/content/journal/micro/10.1099/mic.0.033407-0
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