Localization and assembly of proteins comprising the outer structures of the spore Free

Abstract

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In , however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of ) and IunH (similar to an inosine–uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB–GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB–GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB–GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH–GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB–GFP and IunH–GFP resides in the region between the coat and the exosporium, called the interspace.

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2009-04-01
2024-03-29
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References

  1. Alibek K. 1999 Biohazard New York: Random House;
  2. Aronson A. I., Fitz-James P. 1976; Structure and morphogenesis of the bacterial spore coat. Bacteriol Rev 40:360–402
    [Google Scholar]
  3. Basu S., Kang T. J., Chen W. H., Fenton M. J., Baillie L., Hibbs S., Cross A. S. 2007; Role of Bacillus anthracis spore structures in macrophage cytokine responses. Infect Immun 75:2351–2358
    [Google Scholar]
  4. Bauer T., Little S., Stöver A. G., Driks A. 1999; Functional regions of the Bacillus subtilis spore coat morphogenetic protein CotE. J Bacteriol 181:7043–7051
    [Google Scholar]
  5. Boydston J. A., Chen P., Steichen C. T., Turnbough C. L. Jr 2005; Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis . J Bacteriol 187:5310–5317
    [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. 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, fibroblast, and endothelial cells but not macrophages. Infect Immun 75:4498–4505
    [Google Scholar]
  10. Brahmbhatt T. N., Janes B. K., Stibitz E. S., Darnell S. C., Sanz P., Rasmussen S. B., O'Brien A. D. 2007; Bacillus anthracis exosporium protein BclA affects spore germination, interaction with extracellular matrix proteins, and hydrophobicity. Infect Immun 75:5233–5239
    [Google Scholar]
  11. Brossier F., Levy M., Mock M. 2002; Anthrax spores make an essential contribution to vaccine efficacy. Infect Immun 70:661–664
    [Google Scholar]
  12. Catalano F. A., Meador-Parton J., Popham D. L., Driks A. 2001; Amino acids in the Bacillus subtilis morphogenetic protein SpoIVA with roles in spore coat and cortex formation. J Bacteriol 183:1645–1654
    [Google Scholar]
  13. Chada V. G., Sanstad E. A., Wang R., Driks A. 2003; Morphogenesis of Bacillus spore surfaces. J Bacteriol 185:6255–6261
    [Google Scholar]
  14. Cown W. B., Kethley T. W., Fincher E. L. 1957; The critical-orifice liquid impinger as a sampler for bacterial aerosols. Appl Microbiol 5:119–124
    [Google Scholar]
  15. Cybulski R. J. Jr, Sanz P., McDaniel D., Darnell S., Bull R. L., O'Brien A. D. 2008; Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen. Vaccine 26:4927–4939
    [Google Scholar]
  16. Dowd M. M., Orsburn B., Popham D. L. 2008; Cortex peptidoglycan lytic activity in germinating Bacillus anthracis spores. J Bacteriol 190:4541–4548
    [Google Scholar]
  17. Eichenberger P., Jensen S. T., Conlon E. M., van Ooij C., Silvaggi J., González-Pastor J. E., Fujita M., Ben-Yehuda S., Stragier P. other authors 2003; The σ E regulon and the identification of additional sporulation genes in Bacillus subtilis . J Mol Biol 327:945–972
    [Google Scholar]
  18. Enkhtuya J., Kawamoto K., Kobayashi Y., Uchida I., Rana N., Makino S. 2006; Significant passive protective effect against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids. Microbiology 152:3103–3110
    [Google Scholar]
  19. Fotiadis D., Scheuring S., Muller S. A., Engel A., Muller D. J. 2002; Imaging and manipulation of biological structures with the AFM. Micron 33:385–397
    [Google Scholar]
  20. Fox A., Stewart G. C., Wallera L. N., Fox K. F., Harley W. M., Price R. L. 2003; Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection. J Microbiol Methods 54:143–152
    [Google Scholar]
  21. Friedlander A. M., Welkos S. L., Pitt M. L., Ezzell J. W., Worsham P. L., Rose K. J., Ivins B. E., Lowe J. R., Howe G. B. & other authors; 1993; Postexposure prophylaxis against experimental inhalation anthrax. J Infect Dis 167:1239–1243
    [Google Scholar]
  22. Fritze D. 2004; Taxonomy of the genus Bacillus and related genera: the aerobic endospore-forming bacteria. Phytopathology 94:1245–1248
    [Google Scholar]
  23. 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 coat. J Bacteriol 189:691–705
    [Google Scholar]
  24. Hahn U. K., Boehm R., Beyer W. 2006; DNA vaccination against anthrax in mice – combination of anti-spore and anti-toxin components. Vaccine 24:4569–4571
    [Google Scholar]
  25. Harwood C. R., Cutting S. M. 1990 Molecular Biological Methods for Bacillus Chichester, UK: John Wiley;
  26. Holt S. C., Leadbetter E. R. 1969; Comparative ultrastructure of selected aerobic spore-forming bacteria: a freeze-etching study. Bacteriol Rev 33:346–378
    [Google Scholar]
  27. Institute of Laboratory Animal Resources, Commission on Life Sciences, & National Research Council 1996 Guide for the Care and Use of Laboratory Animals Washington, DC: National Academy Press;
  28. Ivins B. E., Welkos S. L., Knudson G. B., Little S. F. 1990; Immunization against anthrax with aromatic compound-dependent (Aro) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen. Infect Immun 58:303–308
    [Google Scholar]
  29. Ivins B. E., Fellows P. F., Nelson G. O. 1994; Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12:872–874
    [Google Scholar]
  30. Kim H., Hahn M., Grabowski P., McPherson D. C., Wang R., Ferguson C., Eichenberger P., Driks A. 2006; The Bacillus subtilis spore coat protein interaction network. Mol Microbiol 59:487–502
    [Google Scholar]
  31. Koch R. 1876; The etiology of anthrax, based on the life history of Bacillus anthracis . Beitr Biol Pflanz 2:277–310
    [Google Scholar]
  32. Koehler T. M., Dai Z., Kaufman-Yarbray M. 1994; Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans -acting element activate transcription from one of two promoters. J Bacteriol 176:586–595
    [Google Scholar]
  33. La Duc M. T., Satomi M., Venkateswaran K. 2004; Bacillus odysseyi sp. nov., a round-spore-forming bacillus isolated from the Mars Odyssey spacecraft. Int J Syst Evol Microbiol 54:195–201
    [Google Scholar]
  34. Lai E.-M., Phadke N. D., Kachman M. T., Giorno R. S. V., Vazquez J. A., Maddock J. R., Driks A. 2003; Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis . J Bacteriol 185:1443–1454
    [Google Scholar]
  35. Leighton T. J., Doi R. H. 1971; The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis . J Biol Chem 246:3189–3195
    [Google Scholar]
  36. Lemon K. P., Grossman A. D. 1998; Localization of bacterial DNA polymerase: evidence for a factory model of replication. Science 282:1516–1519
    [Google Scholar]
  37. 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]
  38. Losick R., Youngman P., Piggot P. J. 1986; Genetics of endospore formation in Bacillus subtilis . Annu Rev Genet 20:625–669
    [Google Scholar]
  39. 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]
  40. Mann H. B., Whitney D. R. 1947; On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 18:50–60
    [Google Scholar]
  41. Matz L. L., Beaman T. C., Gerhardt P. 1970; Chemical composition of exosporium from spores of Bacillus cereus . J Bacteriol 101:196–201
    [Google Scholar]
  42. May K. R. 1973; The collision nebulizer, description, performance, and applications. J Aerosol Sci 4:235–243
    [Google Scholar]
  43. McPherson D. C., Kim H., Hahn M., Wang R., Grabowski P., Eichenberger P., Driks A. 2005; Characterization of the Bacillus subtilis spore coat morphogenetic protein CotO. J Bacteriol 187:8278–8290
    [Google Scholar]
  44. 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]
  45. Mock M., Fouet A. 2001; Anthrax. Annu Rev Microbiol 55:647–671
    [Google Scholar]
  46. Moir A., Corfe B. M., Behravan J. 2002; Spore germination. Cell Mol Life Sci 59:403–409
    [Google Scholar]
  47. Nicholson W. L. 2002; Roles of Bacillus endospores in the environment. Cell Mol Life Sci 59:410–416
    [Google Scholar]
  48. Ohye D. F., Murrell W. G. 1973; Exosporium and spore coat formation in Bacillus cereus T. J Bacteriol 115:1179–1190
    [Google Scholar]
  49. Oliva C. R., Swiecki M. K., Griguer C. E., Lisanby M. W., Bullard D. C., Turnbough C. L. Jr, Kearney J. F. 2008; The integrin Mac-1 (CR3) mediates internalization and directs Bacillus anthracis spores into professional phagocytes. Proc Natl Acad Sci U S A 105:1261–1266
    [Google Scholar]
  50. 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]
  51. Plomp M., Leighton T., Wheeler K. E., Malkin A. J. 2004; The high-resolution architecture and structural dynamics of Bacillus spores. Biophys J 88:603–608
    [Google Scholar]
  52. Pogliano K., Harry E., Losick R. 1995; Visualization of the subcellular location of sporulation proteins in Bacillus subtilis using immunofluorescence microscopy. Mol Microbiol 18:459–470
    [Google Scholar]
  53. 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]
  54. 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]
  55. Rety S., Salamitou S., Garcia-Verdugo I., Hulmes D. J., Le Hegarat F., Chaby R., Lewit-Bentley A. 2005; The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. J Biol Chem 280:43073–43078
    [Google Scholar]
  56. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  57. 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]
  58. Shao Z., Mou J., Czajkowsky D. M., Yang J., Yuan J. Y. 1996; Biological atomic force microscopy: what is achieved and what is needed. Adv Phys 45:1–86
    [Google Scholar]
  59. Shatalin K. Y., Neyfakh A. A. 2005; Efficient gene inactivation in Bacillus anthracis . FEMS Microbiol Lett 245:315–319
    [Google Scholar]
  60. 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]
  61. 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]
  62. Steichen C. T., Kearney J. F., Turnbough C. L. Jr 2007; Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth. Mol Microbiol 64:359–367
    [Google Scholar]
  63. Sylvestre P., Couture-Tosi E., Mock M. 2002; A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium. Mol Microbiol 45:169–178
    [Google Scholar]
  64. 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]
  65. 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]
  66. Thompson B. M., Waller L. N., Fox K. F., Fox A., Stewart G. C. 2007; The BclB glycoprotein of Bacillus anthracis is involved in exosporium integrity. J Bacteriol 189:6704–6713
    [Google Scholar]
  67. Thorne C. B. 1968; Transduction in Bacillus cereus and Bacillus anthracis . Bacteriol Rev 32:358–361
    [Google Scholar]
  68. 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]
  69. van Ooij C., Eichenberger P., Losick R. 2004; Dynamic patterns of subcellular protein localization during spore coat morphogenesis in Bacillus subtilis . J Bacteriol 186:4441–4448
    [Google Scholar]
  70. Vary P. S. 1994; Prime time for Bacillus megaterium . Microbiology 140:1001–1013
    [Google Scholar]
  71. Waller L. N., Stump M. J., Fox K. F., Harley W. M., Fox A., Stewart G. C., Shahgholi M. 2005; Identification of a second collagen-like glycoprotein produced by Bacillus anthracis and demonstration of associated spore-specific sugars. J Bacteriol 187:4592–4597
    [Google Scholar]
  72. Wang R., Krishnamurthy S. N., Jeong J. S., Driks A., Mehta M., Gingras B. A. 2007; Fingerprinting species and strains of Bacilli spores by distinctive coat surface morphology. Langmuir 23:10230–10234
    [Google Scholar]
  73. Warth A. D., Ohye D. F., Murrell W. G. 1963; The composition and structure of bacterial spores. J Cell Biol 16:579–592
    [Google Scholar]
  74. Weaver J., Kang T. J., Raines K. W., Cao G. L., Hibbs S., Tsai P., Baillie L., Rosen G. M., Cross A. S. 2007; Protective role of Bacillus anthracis exosporium in macrophage-mediated killing by nitric oxide. Infect Immun 75:3894–3901
    [Google Scholar]
  75. 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]
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