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. 1982 Jan 15;202(1):231–241. doi: 10.1042/bj2020231

Characterization, purification and synthesis of spore-coat protein in Bacillus megaterium KM.

G S Stewart, D J Ellar
PMCID: PMC1158096  PMID: 6805468

Abstract

The spore-coat fraction from Bacillus megaterium KM, when prepared by extraction of lysozyme-digested integuments with SDS (sodium dodecyl sulphate) and urea, contains three N-terminal residues and a major component of apparent mol.wt. 17500. Electron microscopy of this fraction shows it to consist of an ordered multilamellar structure similar to that which forms the coat region of intact spores. The 17500-dalton protein, which has been purified to homogeneity, has an N-terminal methionine residue, has high contents of glycine, proline, cysteine and acidic amino acids and readily polymerized even in the presence of thiol-reducing agents. It is first synthesized between late Stage IV and early Stage V, which correlates with the morphological appearance of spore coat. Before Stage VI the 17500-dalton protein is extractable from sporangia by SDS in the absence of thiol-reducing reagents. Between Stage VI and release of mature spores the protein becomes resistant to extraction by SDS unless it is supplemented by a thiol-reducing reagent. In addition to that of the spore-coat protein, the timing of synthesis of all the integument proteins was analysed by SDS/polyacrylamide-gel electrophoresis and non-equilibrium pH-gradient electrophoresis. Several integument proteins are conservatively synthesized from as early as 1h after the end of exponential growth (t1), which may reflect protein incorporation into the spore outer membrane.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. Aronson A. I., Fitz-James P. C. Biosynthesis of bacterial spore coats. J Mol Biol. 1968 Apr 14;33(1):199–212. doi: 10.1016/0022-2836(68)90288-x. [DOI] [PubMed] [Google Scholar]
  3. Aronson A. I., Fitz-James P. Structure and morphogenesis of the bacterial spore coat. Bacteriol Rev. 1976 Jun;40(2):360–402. doi: 10.1128/br.40.2.360-402.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bulla L. A., Jr, Bechtel D. B., Kramer K. J., Shethna Y. I., Aronson A. I., Fitz-James P. C. Ultrastructure, physiology, and biochemistry of Bacillus thuringiensis. Crit Rev Microbiol. 1980;8(2):147–204. doi: 10.3109/10408418009081124. [DOI] [PubMed] [Google Scholar]
  5. Cassier M., Ryter A. Sur un mutant de Clostridium perfringens donnant des spores sans tuniques à germination lysozyme-dépendante. Ann Inst Pasteur (Paris) 1971 Dec;121(6):717–732. [PubMed] [Google Scholar]
  6. Cassier M., Sebald M. Germination lysozyme-dépendante des spores de Clostridium perfringens ATCC 3624 après traitement thermique. Ann Inst Pasteur (Paris) 1969 Sep;117(3):312–324. [PubMed] [Google Scholar]
  7. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  8. Cheng Y. S., Aronson A. I. Alterations of spore coat processing and protein turnover in a Bacillus cereus mutant with a defective postexponential intracellular protease. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1254–1258. doi: 10.1073/pnas.74.3.1254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crafts-Lighty A., Ellar D. J. The structure and function of the spore outer membrane in dormant and germinating spores of Bacillus megaterium. J Appl Bacteriol. 1980 Feb;48(1):135–145. doi: 10.1111/j.1365-2672.1980.tb05215.x. [DOI] [PubMed] [Google Scholar]
  10. DiRienzo J. M., Inouye M. Lipid fluidity-dependent biosynthesis and assembly of the outer membrane proteins of E. coli. Cell. 1979 May;17(1):155–161. doi: 10.1016/0092-8674(79)90303-9. [DOI] [PubMed] [Google Scholar]
  11. GERHARDT P., BLACK S. H. Permeability of bacterial spores. II. Molecular variables affecting solute permeation. J Bacteriol. 1961 Nov;82:750–760. doi: 10.1128/jb.82.5.750-760.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldman R. C., Tipper D. J. Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component. J Bacteriol. 1978 Sep;135(3):1091–1106. doi: 10.1128/jb.135.3.1091-1106.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  15. Linn T., Losick R. The program of protein synthesis during sporulation in Bacillus subtilis. Cell. 1976 May;8(1):103–114. doi: 10.1016/0092-8674(76)90191-4. [DOI] [PubMed] [Google Scholar]
  16. Munoz L., Sadaie Y., Doi R. H. Spore coat protein of Bacillus subtilis. Structure and precursor synthesis. J Biol Chem. 1978 Oct 10;253(19):6694–6701. [PubMed] [Google Scholar]
  17. Nakayama T., Munoz L. E., Sadaie Y., Doi R. H. Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis. J Bacteriol. 1978 Sep;135(3):952–960. doi: 10.1128/jb.135.3.952-960.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  19. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  20. Scott I. R., Ellar D. J. Metabolism and the triggering of germination of Bacillus megaterium. Use of L-[3H]alanine and tritiated water to detect metabolism. Biochem J. 1978 Aug 15;174(2):635–640. doi: 10.1042/bj1740635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stelma G. N., Jr, Aronson A. I., Fitz-James P. C. A Bacillus cereus mutant defective in spore coat deposition. J Gen Microbiol. 1980 Jan;116(1):173–185. doi: 10.1099/00221287-116-1-173. [DOI] [PubMed] [Google Scholar]
  22. Stewart G. S., Eaton M. W., Johnstone K., Barrett M. D., Ellar D. J. An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy. Biochim Biophys Acta. 1980 Aug 4;600(2):270–290. doi: 10.1016/0005-2736(80)90432-0. [DOI] [PubMed] [Google Scholar]
  23. Stewart G. S., Johnstone K., Hagelberg E., Ellar D. J. Commitment of bacterial spores to germinate. A measure of the trigger reaction. Biochem J. 1981 Jul 15;198(1):101–106. doi: 10.1042/bj1980101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suzuki Y., Rode L. J. Effect of lysozyme on resting spores of Bacillus megaterium. J Bacteriol. 1969 Apr;98(1):238–245. doi: 10.1128/jb.98.1.238-245.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]

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