1887

Abstract

Germination of endospores of involves the activities of several germination-specific lytic enzymes, including glucosaminidase and lytic transglycosylase. Another non-hydrolytic activity, likely to be due to an epimerase, also occurs. The effect of pH on enzyme activities and the overall germination rate was measured. Optimal germination occurred between pH 7–9; however, optimum glucosaminidase and epimerase activities were noted at pH 5. Conversely, the lytic transglycosylase activity was greatest at pH 8. Treatment of spores (15 min) with heat (90 °C) or NaOH (025 M) led to impaired cortex hydrolysis/modification, but with <20% loss in viability. Analysis of muropeptides in the germination exudate revealed a reduction of >85% in glucosaminidase and epimerase products, when compared to untreated spores. Conversely, lytic transglycosylase activity was increased by alkali or heat treatment, which was possibly due to increased substrate availability. FB101 () spores, which lack lytic transglycosylase activity, showed 90-fold greater loss in viability than the wild-type after 1 h at 90 °C. Similarly, 97% of FB101 () spores were unable to form a colony on nutrient agar after 130 min exposure to 025 M NaOH at 4 °C, whereas the wild-type was unaffected. This demonstrates the crucial role of the lytic transglycosylase in cortex hydrolysis of damaged spores. The respective targets of heat and alkali in spores and their role during germination are discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-11-2925
2001-11-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/11/1472925a.html?itemId=/content/journal/micro/10.1099/00221287-147-11-2925&mimeType=html&fmt=ahah

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). Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species. J Appl Microbiol 91, 1-9.[CrossRef] [Google Scholar]
  3. Atrih, A., Zöllner, 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., Zöllner, P., Allmaier, G., Williamson, M. P. & 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., Körner, 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. Belliveau, B. H., Beaman, T. C., Pankratz, S. & Gerhardt, P. (1992). Heat killing of bacterial spores analysed by differential scanning calorimetry. J Bacteriol 174, 4463-4474. [Google Scholar]
  7. Boland, F. M., 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., Miyata, S., Makino, S. & Moriyama, R. (1997). Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene. J Bacteriol 179, 3181-3187. [Google Scholar]
  9. Chen, Y., Fukuoka, S. & Makino, S. (2000). A novel spore peptidoglycan hydrolase of Bacillus cereus, biochemical characterization and nucleotide sequence of the corresponding gene, sleL. J Bacteriol 182, 1499-1506.[CrossRef] [Google Scholar]
  10. Ciarciaglini, G., Hill, P. J., Davies, K., MacClure, P. J., Kilsby, D., Brown, M. H. & Coote, P. J. (2000). Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores. Appl Environ Microbiol 66, 3735-3742.[CrossRef] [Google Scholar]
  11. Duncan, C. L., Labbe, R. G. & Reich, R. R. (1972). Germination of heat- and alkali-altered spores of Clostridium perfringens type A by lysozyme and an initiation protein. J Bacteriol 109, 550-559. [Google Scholar]
  12. Ellar, D. J. (1978). Spore specific structures and their functions. In Relations between Structure and Function in the Prokaryotic Cell (Society for General Microbiology Symposium no. 28) , pp. 295-325. Edited by R. Y. Stanier, H. J. Rogers & J. B. Ward. Cambridge:Cambridge University Press.
  13. 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]
  14. Foster, S. J. & Johnstone, K. (1988). Germination-specific cortex-lytic enzyme is activated during triggering of Bacillus megaterium KM spore germination. Mol Microbiol 2, 727-733.[CrossRef] [Google Scholar]
  15. Foster, S. J. & Johnstone, K. (1990). Pulling the trigger, the mechanism of bacterial spore germination. Mol Microbiol 4, 137-141.[CrossRef] [Google Scholar]
  16. Gerhardt, P. & Marquis, R. E. (1989). Spore thermoresistance mechanisms. In Regulation of Procaryotic Development , pp. 43-63. Edited by I. Smith, R. Slepecky & P. Setlow. Washington, DC:American Society for Microbiology.
  17. Gould, G. W., Stubbs, J. M. & King, W. L. (1970). Structure composition and resistant layers in bacterial spore coats. J Gen Microbiol 60, 347-355.[CrossRef] [Google Scholar]
  18. Ishikawa, S., Yamane, K. & Sekiguchi, J. (1998). Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores. J Bacteriol 180, 1375-1380. [Google Scholar]
  19. Labbe, R. G. & Chang, C. A. (1995). Recovery of heat-injured spores of Clostridium perfringens type B, C and D by lysozyme and an initiation protein. Lett Appl Microbiol 21, 302-306.[CrossRef] [Google Scholar]
  20. 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]
  21. Marquis, R. E., Sim, J. & Shin, S. Y. (1994). Molecular mechanism of resistance to heat and oxidative damage. J Appl Bacteriol 76, 40-48.[CrossRef] [Google Scholar]
  22. Miyata, S., Moriyama, R., Sogimoto, K. & Makino, S. (1995). Purification and partial characterization of a spore cortex-lytic enzyme of Clostridium perfringens S40 spores. Biosci Biotechnol Biochem 59, 514-515.[CrossRef] [Google Scholar]
  23. Moriyama, R., Kudoh, S., Miyata, S., Nonobe, S., Hattori, A. & Makino, S. (1996). A germination-specific spore cortex enzyme from Bacillus cereus spores, cloning and sequencing of the gene and molecular characterization of the enzyme. J Bacteriol 178, 5330-5332. [Google Scholar]
  24. 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 localization on the exterior side of the cortex in dormant spores. J Bacteriol 181, 2373-2378. [Google Scholar]
  25. Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J. & Setlow, P. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64, 548-572.[CrossRef] [Google Scholar]
  26. Oloyede, O. B. & Scholefield, J. (1994). Inhibition of Bacillus spores by combinations of heat, potassium sorbate, NaCl and pH. World J Microbiol Biotechnol 10, 579-582.[CrossRef] [Google Scholar]
  27. Paidhungat, M. & Setlow, P. (2000). Role of Ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. J Bacteriol 182, 2513-2519.[CrossRef] [Google Scholar]
  28. Popham, D. L., Hanlin, 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. Sekiguchi, J., Akeo, K., Yamamoto, H., Khasanov, F. K., Alonso, J. C. & Kuroda, A. (1995). Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. J Bacteriol 177, 5582-5589. [Google Scholar]
  30. Stewart, G. S. A. B., Johnstone, K., Hagelberg, E. & Ellar, D. J. (1981). Commitment of bacterial spores to germinate. Biochem J 198, 101-106. [Google Scholar]
  31. Warth, A. D. (1978). Molecular structure of the bacterial spore. Adv Microb Physiol 17, 1-47. [Google Scholar]
  32. Warth, A. D. (1980). Heat stability of Bacillus cereus enzymes within spores and in extracts. J Bacteriol 143, 27-34. [Google Scholar]
  33. Wax, R. & Freese, E. (1968). Initiation of the germination of Bacillus subtilis spores by a combination of compounds in place of l-alanine. J Bacteriol 95, 433-438. [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-11-2925
Loading
/content/journal/micro/10.1099/00221287-147-11-2925
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error