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Volume 148, Issue 8

Analysis of spore cortex lytic enzymes and related proteins in endospore germination Free

Abstract

The location and function of recognized cortex-lytic enzymes of have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The gene is the second gene in a bicistronic operon with . The SleB protein is absent from mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB – in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme’s hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore’s ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of . Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination: the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

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Keyword(s): germination-specific cortex-lytic enzymes , GSLE, germination-specific cortex-lytic enzyme , RP-HPLC, reverse-phase HPLC , yaaH and ypeB
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2002-08-01
2024-04-19
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References

  1. Atrih A., Foster S. J. 1999; The role of peptidoglycan structure and structural dynamics during endospore dormancy and germination. Antonie Leeuwenhoek 75:299–307 [CrossRef]
     [Google Scholar]
  2. Atrih A., Foster S. J. 2001; In vivo roles of the germination-specific lytic enzymes of Bacillus subtilis 168. Microbiology 147:57–64
     [Google Scholar]
  3. Atrih A., Zollner P., Allmaier G., Foster S. J. 1996; Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation. J Bacteriol 178:6173–6183
     [Google Scholar]
  4. Atrih A., Zollner P., Allmaier G., Williamson M., Foster S. J. 1998; Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores. J Bacteriol 180:4603–4612
     [Google Scholar]
  5. Atrih A., Bacher G., Korner R., Allmaier G., Foster S. J. 1999; Structural analysis of Bacillus megaterium KM spore peptidoglycan and its dynamics during germination. Microbiology 145:1033–1041 [CrossRef]
     [Google Scholar]
  6. Bagyan I., Noback M., Bron S., Paidhungat M., Setlow P. 1998; Characterisation of yhcN , a new forespore-specific gene of Bacillus subtilis . Gene 212:179–188 [CrossRef]
     [Google Scholar]
  7. Boland F., Atrih A., Chirakkal H., Foster S. J., Moir A. 2000; Complete spore cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB. Microbiology 146:57–64
     [Google Scholar]
  8. Chen Y., Fukuoka S., Makino S. 2000a; A novel spore peptidoglycan hydrolase of Bacillus cereus : biochemical characterisation and nucleotide sequence of the corresponding gene, sleL . J Bacteriol 182:1499–1506 [CrossRef]
     [Google Scholar]
  9. Chen Y., Miyata S., Makino S., Moriyama R. 2000b; Molecular characterisation of a germination specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene. J Bacteriol 197:3181–3187
     [Google Scholar]
  10. Foster S. J., Johnstone K. 1987; Purification and properties of a germination-specific cortex lytic enzyme from spores of Bacillus megaterium KM. Biochem J 242:573–579
     [Google Scholar]
  11. Guerout-Fleury A., Shazand K., Frandsen N., Stragier P. 1995; Antibiotic resistance cassettes for Bacillus subtilis . Gene 167:335–336 [CrossRef]
     [Google Scholar]
  12. Horsburgh M. J., Moir A. 1999; Sigma M, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt. Mol Microbiol 32:41–50 [CrossRef]
     [Google Scholar]
  13. Hudson K. D., Corfe B. M., Kemp E. H., Feavers I. M., Coote P. J., Moir A. 2001; Localization of GerAA and GerAC proteins in the Bacillus subtilis spore. J Bacteriol 183:4317–4322 [CrossRef]
     [Google Scholar]
  14. Ishikawa S., Yamane K., Sekiguchi J. 1998; Regulation and characterisation of a newly deduced cell wall hydrolase gene ( cwlJ ) which affects germination of Bacillus subtilis spores. J Bacteriol 180:1375–1380
     [Google Scholar]
  15. Kodama T., Takamatsu H., Asai K., Kobayashi K., Ogasawara N., Watabe K. 1999; The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores. J Bacteriol 181:4584–4591
     [Google Scholar]
  16. Kodama T., Takamatsu H., Asai K., Sadie Y., Watabe K. 2000; Synthesis and characterisation of the spore proteins of Bacillus subtilis YdhD, YkuD and YkvP, which carry a motif conserved among cell wall binding proteins. J Biochem 128:655–663 [CrossRef]
     [Google Scholar]
  17. Kunst F., Rapoport G. 1995; Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis . J Bacteriol 177:2403–2407
     [Google Scholar]
  18. Makino S., Ito N., Inoue T., Miyata S., Moriyama R. 1994; A spore-lytic enzyme released from Bacillus cereus spores during germination. Microbiology 140:1403–1410 [CrossRef]
     [Google Scholar]
  19. Miyata S., Moriyama R., Miyahara N., Makino S. 1995; A gene ( sleC ) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterisation. Microbiology 141:2643–2650 [CrossRef]
     [Google Scholar]
  20. Moir A., Corfe B. M., Behravan J. 2002; Spore germination. Cell Mol Life Sci 59:403–409 [CrossRef]
     [Google Scholar]
  21. Moriyama R., Hattori A., Miyata S., Kudoh S., Makino S. 1996a; A gene ( sleB ) encoding a spore cortex lytic enzyme from Bacillus subtilis and its response to l-alanine mediated germination. J Bacteriol 178:6059–6063
     [Google Scholar]
  22. Moriyama R., Kudoh S., Miyata S., Nonobe S., Hattori A., Makino S. 1996b; A germination specific cortex lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterisation of the enzyme. J Bacteriol 178:5330–5332
     [Google Scholar]
  23. Moriyama R., Fukuoka H., Miyata S., Kudoh S., Hattori A., Kozuka S., Yasuda Y., Tochikubo K., Makino S. 1999; Expression of a germination specific amidase, SleB, of bacilli in the forespore compartment of sporulating cells and its localisation on the exterior side of the cortex in dormant spores. J Bacteriol 181:2373–2378
     [Google Scholar]
  24. Paidhungat M., Setlow P. 1999; Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant d-alanine. J Bacteriol 181:3341–3350
     [Google Scholar]
  25. Paidhungat M., Setlow P. 2000; Role of Ger proteins in nutrient and non-nutrient triggering of spore germination in Bacillus subtilis . J Bacteriol 182:2513–2519 [CrossRef]
     [Google Scholar]
  26. Paidhungat M., Setlow P. 2001; Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J Bacteriol 183:3982–3990 [CrossRef]
     [Google Scholar]
  27. Paidhungat M., Ragkousi K., Setlow P. 2001; Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. J Bacteriol 183:4886–4893 [CrossRef]
     [Google Scholar]
  28. Popham D. J., Helin J., Costello C. E., Setlow P. 1996; Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci USA 93:15405–15410 [CrossRef]
     [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  30. Southworth T. W., Guffanti A. A., Moir A., Krulwich T. A. 2001; GerN: an endospore germination protein of Bacillus cereus is a Na+/H+-K+ antiporter. J Bacteriol 183:5896–5903 [CrossRef]
     [Google Scholar]
  31. Stewart G. S. A. B., Johnstone K., Hagelberg E., Ellar D. 1981; Commitment of bacterial spores to germinate. Biochem J 198:101–106
     [Google Scholar]
  32. Thackray P. D., Behravan J., Southworth T. W., Moir A. 2001; GerN, an antiporter homologue important in the germination of Bacillus cereus endospores. J Bacteriol 183:476–482 [CrossRef]
     [Google Scholar]
  33. Vagner V., Dervyn E., Ehrlich S. D. 1998; A vector for systematic gene disruption in Bacillus subtilis . Microbiology 144:3097–3104 [CrossRef]
     [Google Scholar]
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Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination
Microbiology 148, 2383 (2002); https://doi.org/10.1099/00221287-148-8-2383
/content/journal/micro/10.1099/00221287-148-8-2383
/content/journal/micro/10.1099/00221287-148-8-2383
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