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. 1995 May;177(9):2481–2489. doi: 10.1128/jb.177.9.2481-2489.1995

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1.

J W Farchaus 1, W J Ribot 1, M B Downs 1, J W Ezzell 1
PMCID: PMC176908  PMID: 7730281

Abstract

Many prokaryotic organisms possess surface layer (S-layer) proteins that are components of the outermost cell envelope. With immunogold labeling, it was demonstrated that the protein extractable antigen 1 (EA1) was localized on the outer surface and specifically to cell wall fragments from Bacillus anthracis which retained the S layer. When grown in rich medium under aerobic conditions, the avirulent strain Delta Sterne-1 released large amounts of EA1 into the medium. This EA1 had no higher-order structure initially but formed two-dimensional crystals under defined conditions. The released EA1 was purified in aqueous buffers with a three-step procedure and found to have a mass of 95 kDa when subjected to denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence data revealed exact identity to the first eight residues of the S-layer protein from B. thuringiensis 4045. Gel permeation chromatography of the purified EA1 under nondenaturing conditions revealed a single peak corresponding to a mass of approximately 400 kDa, suggesting that a tetramer or dimer of dimers was the primary species in solution. SDS-PAGE of EA1 purified in the absence of protease inhibitors revealed specific proteolytic processing to an 80-kDa form, which immunoreacted with polyclonal anti-EA1 antibodies. This proteolytic cleavage of EA1 to 80 kDa was duplicated with purified EA1 and the protease trypsin or pronase.

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Selected References

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  1. Adachi T., Yamagata H., Tsukagoshi N., Udaka S. Repression of the cell wall protein gene operon in Bacillus brevis 47 by magnesium and calcium ions. J Bacteriol. 1991 Jul;173(13):4243–4245. doi: 10.1128/jb.173.13.4243-4245.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrantes F. J. The nicotinic cholinergic receptor : different compositions evidenced by statistical analysis. Biochem Biophys Res Commun. 1975 Jan 20;62(2):407–414. doi: 10.1016/s0006-291x(75)80153-7. [DOI] [PubMed] [Google Scholar]
  3. Dubreuil J. D., Kostrzynska M., Austin J. W., Trust T. J. Antigenic differences among Campylobacter fetus S-layer proteins. J Bacteriol. 1990 Sep;172(9):5035–5043. doi: 10.1128/jb.172.9.5035-5043.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ezzell J. W., Jr, Abshire T. G. Immunological analysis of cell-associated antigens of Bacillus anthracis. Infect Immun. 1988 Feb;56(2):349–356. doi: 10.1128/iai.56.2.349-356.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ezzell J. W., Jr, Abshire T. G., Little S. F., Lidgerding B. C., Brown C. Identification of Bacillus anthracis by using monoclonal antibody to cell wall galactose-N-acetylglucosamine polysaccharide. J Clin Microbiol. 1990 Feb;28(2):223–231. doi: 10.1128/jcm.28.2.223-231.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gerhardt P. Cytology of Bacillus anthracis. Fed Proc. 1967 Sep;26(5):1504–1517. [PubMed] [Google Scholar]
  7. Green B. D., Battisti L., Koehler T. M., Thorne C. B., Ivins B. E. Demonstration of a capsule plasmid in Bacillus anthracis. Infect Immun. 1985 Aug;49(2):291–297. doi: 10.1128/iai.49.2.291-297.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hastie A. T., Brinton C. C., Jr Isolation, characterization, and in vitro assembly of the tetragonally arrayed layer of Bacillus sphaericus. J Bacteriol. 1979 Jun;138(3):999–1009. doi: 10.1128/jb.138.3.999-1009.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hastie A. T., Brinton C. C., Jr Specific interaction of the tetragonally arrayed protein layer of Bacillus sphaericus with its peptidoglycan sacculus. J Bacteriol. 1979 Jun;138(3):1010–1021. doi: 10.1128/jb.138.3.1010-1021.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Holt S. C., Leadbetter E. R. Comparative ultrastructure of selected aerobic spore-forming bacteria: a freeze-etching study. Bacteriol Rev. 1969 Jun;33(2):346–378. doi: 10.1128/br.33.2.346-378.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hovmöller S., Sjögren A., Wang D. N. The structure of crystalline bacterial surface layers. Prog Biophys Mol Biol. 1988;51(2):131–163. doi: 10.1016/0079-6107(88)90012-0. [DOI] [PubMed] [Google Scholar]
  12. Ivins B. E., Ezzell J. W., Jr, Jemski J., Hedlund K. W., Ristroph J. D., Leppla S. H. Immunization studies with attenuated strains of Bacillus anthracis. Infect Immun. 1986 May;52(2):454–458. doi: 10.1128/iai.52.2.454-458.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jermyn M. A. Increasing the sensitivity of the anthrone method for carbohydrate. Anal Biochem. 1975 Sep;68(1):332–335. doi: 10.1016/0003-2697(75)90713-7. [DOI] [PubMed] [Google Scholar]
  14. Leach B. S., Collawn J. F., Jr, Fish W. W. Behavior of glycopolypeptides with empirical molecular weight estimation methods. 1. In sodium dodecyl sulfate. Biochemistry. 1980 Dec 9;19(25):5734–5741. doi: 10.1021/bi00566a011. [DOI] [PubMed] [Google Scholar]
  15. Luckevich M. D., Beveridge T. J. Characterization of a dynamic S layer on Bacillus thuringiensis. J Bacteriol. 1989 Dec;171(12):6656–6667. doi: 10.1128/jb.171.12.6656-6667.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Makino S., Sasakawa C., Uchida I., Terakado N., Yoshikawa M. Cloning and CO2-dependent expression of the genetic region for encapsulation from Bacillus anthracis. Mol Microbiol. 1988 May;2(3):371–376. doi: 10.1111/j.1365-2958.1988.tb00041.x. [DOI] [PubMed] [Google Scholar]
  17. Makino S., Uchida I., Terakado N., Sasakawa C., Yoshikawa M. Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J Bacteriol. 1989 Feb;171(2):722–730. doi: 10.1128/jb.171.2.722-730.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Messner P., Sleytr U. B. Crystalline bacterial cell-surface layers. Adv Microb Physiol. 1992;33:213–275. doi: 10.1016/s0065-2911(08)60218-0. [DOI] [PubMed] [Google Scholar]
  19. Mikesell P., Ivins B. E., Ristroph J. D., Dreier T. M. Evidence for plasmid-mediated toxin production in Bacillus anthracis. Infect Immun. 1983 Jan;39(1):371–376. doi: 10.1128/iai.39.1.371-376.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mock M., Labruyère E., Glaser P., Danchin A., Ullmann A. Cloning and expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in Escherichia coli. Gene. 1988 Apr 29;64(2):277–284. doi: 10.1016/0378-1119(88)90342-3. [DOI] [PubMed] [Google Scholar]
  21. Ohmizu H., Sasaki T., Tsukagoshi N., Udaka S., Kaneda N., Yagi K. Major proteins released by a protein-producing bacterium, Bacillus brevis 47, are derived from cell wall protein. J Biochem. 1983 Oct;94(4):1077–1084. doi: 10.1093/oxfordjournals.jbchem.a134450. [DOI] [PubMed] [Google Scholar]
  22. Phillips A. P., Ezzell J. W. Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens. J Appl Bacteriol. 1989 May;66(5):419–432. doi: 10.1111/j.1365-2672.1989.tb05111.x. [DOI] [PubMed] [Google Scholar]
  23. Ristroph J. D., Ivins B. E. Elaboration of Bacillus anthracis antigens in a new, defined culture medium. Infect Immun. 1983 Jan;39(1):483–486. doi: 10.1128/iai.39.1.483-486.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robertson D. L., Leppla S. H. Molecular cloning and expression in Escherichia coli of the lethal factor gene of Bacillus anthracis. Gene. 1986;44(1):71–78. doi: 10.1016/0378-1119(86)90044-2. [DOI] [PubMed] [Google Scholar]
  25. Robertson D. L., Tippetts M. T., Leppla S. H. Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase. Gene. 1988 Dec 20;73(2):363–371. doi: 10.1016/0378-1119(88)90501-x. [DOI] [PubMed] [Google Scholar]
  26. Sleytr U. B., Messner P. Crystalline surface layers on bacteria. Annu Rev Microbiol. 1983;37:311–339. doi: 10.1146/annurev.mi.37.100183.001523. [DOI] [PubMed] [Google Scholar]
  27. Sleytr U. B., Messner P., Pum D., Sára M. Crystalline bacterial cell surface layers. Mol Microbiol. 1993 Dec;10(5):911–916. doi: 10.1111/j.1365-2958.1993.tb00962.x. [DOI] [PubMed] [Google Scholar]
  28. Sára M., Pum D., Küpcü S., Messner P., Sleytr U. B. Isolation of two physiologically induced variant strains of Bacillus stearothermophilus NRS 2004/3a and characterization of their S-layer lattices. J Bacteriol. 1994 Feb;176(3):848–860. doi: 10.1128/jb.176.3.848-860.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Uchida I., Sekizaki T., Hashimoto K., Terakado N. Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid. J Gen Microbiol. 1985 Feb;131(2):363–367. doi: 10.1099/00221287-131-2-363. [DOI] [PubMed] [Google Scholar]
  30. Welkos S. L., Lowe J. R., Eden-McCutchan F., Vodkin M., Leppla S. H., Schmidt J. J. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene. 1988 Sep 30;69(2):287–300. doi: 10.1016/0378-1119(88)90439-8. [DOI] [PubMed] [Google Scholar]

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