Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Jun;175(12):3757–3766. doi: 10.1128/jb.175.12.3757-3766.1993

Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis.

J Zhang 1, P C Fitz-James 1, A I Aronson 1
PMCID: PMC204792  PMID: 8509331

Abstract

The Bacillus subtilis spore coat is composed of at least 15 polypeptides plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat protein and is resistant to solubilization by a variety of reagents, implying extensive cross-linking. A dodecapeptide was purified from this fraction by formic acid hydrolysis and reverse-phase high-performance liquid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodecapeptide at its amino end was clustered with four other genes designated cotV, cotW, cotY, and cotZ. These genes were mapped to 107 degrees between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both proteins are cysteine rich, and CotY antigen was present in spore coat extracts as disulfide cross-linked multimers. There was little CotX antigen in the spore coat soluble fraction, and deletion of this gene resulted in a 30% reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotX, cotYZ, or cotXYZ genes were heat and lysozyme resistant but readily clumped and responded more rapidly to germinants than did spores from the wild type. In electron micrographs, there was a less densely staining outer coat in spores produced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These proteins, as part of the coat insoluble fraction, appear to be localized to the outer coat and influence spore hydrophobicity as well as the accessibility of germinants.

Full text

PDF
3757

Images in this article

3763Image
on p.3763
3764Image
on p.3764
3764Image
on p.3764

Selected References

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

  1. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Aronson A. I., Song H. Y., Bourne N. Gene structure and precursor processing of a novel Bacillus subtilis spore coat protein. Mol Microbiol. 1989 Mar;3(3):437–444. doi: 10.1111/j.1365-2958.1989.tb00189.x. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bourne N., FitzJames P. C., Aronson A. I. Structural and germination defects of Bacillus subtilis spores with altered contents of a spore coat protein. J Bacteriol. 1991 Oct;173(20):6618–6625. doi: 10.1128/jb.173.20.6618-6625.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cariello L., Wilson J., Lorand L. Activation of transglutaminase during embryonic development. Biochemistry. 1984 Dec 18;23(26):6843–6850. doi: 10.1021/bi00321a087. [DOI] [PubMed] [Google Scholar]
  7. Dedonder R. A., Lepesant J. A., Lepesant-Kejzlarová J., Billault A., Steinmetz M., Kunst F. Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl Environ Microbiol. 1977 Apr;33(4):989–993. doi: 10.1128/aem.33.4.989-993.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donovan W., Zheng L. B., Sandman K., Losick R. Genes encoding spore coat polypeptides from Bacillus subtilis. J Mol Biol. 1987 Jul 5;196(1):1–10. doi: 10.1016/0022-2836(87)90506-7. [DOI] [PubMed] [Google Scholar]
  9. Eyre D. R. Collagen: molecular diversity in the body's protein scaffold. Science. 1980 Mar 21;207(4437):1315–1322. doi: 10.1126/science.7355290. [DOI] [PubMed] [Google Scholar]
  10. Fitz-James P. C. Formation of protoplasts from resting spores. J Bacteriol. 1971 Mar;105(3):1119–1136. doi: 10.1128/jb.105.3.1119-1136.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Foerder C. A., Shapiro B. M. Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4214–4218. doi: 10.1073/pnas.74.10.4214. [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. Greenberg C. S., Birckbichler P. J., Rice R. H. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 1991 Dec;5(15):3071–3077. doi: 10.1096/fasebj.5.15.1683845. [DOI] [PubMed] [Google Scholar]
  14. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  15. Haima P., Bron S., Venema G. The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg. Mol Gen Genet. 1987 Sep;209(2):335–342. doi: 10.1007/BF00329663. [DOI] [PubMed] [Google Scholar]
  16. Hoch J. A., Barat M., Anagnostopoulos C. Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J Bacteriol. 1967 Jun;93(6):1925–1937. doi: 10.1128/jb.93.6.1925-1937.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Itaya M., Kondo K., Tanaka T. A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. Nucleic Acids Res. 1989 Jun 12;17(11):4410–4410. doi: 10.1093/nar/17.11.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kopan R., Fuchs E. A new look into an old problem: keratins as tools to investigate determination, morphogenesis, and differentiation in skin. Genes Dev. 1989 Jan;3(1):1–15. doi: 10.1101/gad.3.1.1. [DOI] [PubMed] [Google Scholar]
  19. Landon Cleavage at aspartyl-prolyl bonds. Methods Enzymol. 1977;47:145–149. doi: 10.1016/0076-6879(77)47017-4. [DOI] [PubMed] [Google Scholar]
  20. Moir A. Germination properties of a spore coat-defective mutant of Bacillus subtilis. J Bacteriol. 1981 Jun;146(3):1106–1116. doi: 10.1128/jb.146.3.1106-1116.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pandey N. K., Aronson A. I. Properties of the Bacillus subtilis spore coat. J Bacteriol. 1979 Mar;137(3):1208–1218. doi: 10.1128/jb.137.3.1208-1218.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Piggot P. J., Hoch J. A. Revised genetic linkage map of Bacillus subtilis. Microbiol Rev. 1985 Jun;49(2):158–179. doi: 10.1128/mr.49.2.158-179.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  24. Stewart G. S., Ellar D. J. Characterization, purification and synthesis of spore-coat protein in Bacillus megaterium KM. Biochem J. 1982 Jan 15;202(1):231–241. doi: 10.1042/bj2020231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Triglia T., Peterson M. G., Kemp D. J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988 Aug 25;16(16):8186–8186. doi: 10.1093/nar/16.16.8186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wax R., Freese E. Initiation of the germination of Bacillus subtilis spores by a combination of compounds in place of L-alanine. J Bacteriol. 1968 Feb;95(2):433–438. doi: 10.1128/jb.95.2.433-438.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wiencek K. M., Klapes N. A., Foegeding P. M. Hydrophobicity of Bacillus and Clostridium spores. Appl Environ Microbiol. 1990 Sep;56(9):2600–2605. doi: 10.1128/aem.56.9.2600-2605.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zheng L. B., Donovan W. P., Fitz-James P. C., Losick R. Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore. Genes Dev. 1988 Aug;2(8):1047–1054. doi: 10.1101/gad.2.8.1047. [DOI] [PubMed] [Google Scholar]
  29. Zheng L. B., Losick R. Cascade regulation of spore coat gene expression in Bacillus subtilis. J Mol Biol. 1990 Apr 20;212(4):645–660. doi: 10.1016/0022-2836(90)90227-d. [DOI] [PubMed] [Google Scholar]
  30. Zheng L., Halberg R., Roels S., Ichikawa H., Kroos L., Losick R. Sporulation regulatory protein GerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes. J Mol Biol. 1992 Aug 20;226(4):1037–1050. doi: 10.1016/0022-2836(92)91051-p. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES