Characterization and deposition of the proteins in the outermost layer of Bacillus megaterium spore
Related Experiment Videos
Y Takubo1, M Okuda, I Takemura
1Department of Environmental Chemistry and Microbiology, Faculty of Pharmaceutical Sciences, Osaka University.
Abstract:
It was proved that three spore coat proteins of 48, 36, and 22 kDa (P48, P36, and P22) were the components of the outermost layer (OL) of Bacillus megaterium ATCC 12872 spore by analysis of the isolated OL. And it was indicated that these proteins were deposited not by disulfide bond, but by ionic and/or hydrophobic bonds on the spore. Among them, P36 and P22 were expected to be located on the very surface of the spore by immunological analysis. In the OL deficient mutant of B. megaterium ATCC 12872, MAE05, whose spore was lacking in these OL proteins and galactosamine-6-phosphate polymer, both P36 and P22 were present in the mother cell cytoplasm and deposited on the forespores, but they disappeared with the lysis of mother cells. An OL protein-releasing factor having proteolytic activity was detected in the culture supernatant at the late sporulating stage of both the wild-type and the mutant strains. But the factor could not act on the proteins of the mature spores and the forespores at t10 (tn indicates n hr after the end of exponential growth) of the wild-type strain. Moreover, P36 and P22 were found in the spores of a revertant of MAE05 which could form galactosamine-6-phosphate polymer, suggesting that this sugar polymer played the role in protecting the OL proteins against the protease-like substance after the deposition.
Insights
Bacillus megaterium spore coat proteins P36 and P22 are key components of the outermost layer. A protective sugar polymer prevents their degradation by proteases after deposition.
Area of Science:
- Microbiology
- Bacterial spore formation
- Protein biochemistry
Background:
- The outermost layer (OL) of Bacillus megaterium spores plays a crucial role in spore structure and protection.
- Understanding the composition and assembly of the OL is essential for deciphering spore resistance mechanisms.
Purpose of the Study:
- To identify the protein components of the Bacillus megaterium spore outermost layer.
- To elucidate the deposition mechanism and potential protective factors for these proteins.
Main Methods:
- Isolation and analysis of spore outermost layer components.
- Immunological analysis to determine protein localization.
- Characterization of an OL protein-releasing factor with proteolytic activity.
- Analysis of OL-deficient mutants and revertants.
Main Results:
- Three spore coat proteins (P48, P36, P22) were identified as components of the outermost layer, bound by ionic and/or hydrophobic interactions.
- P36 and P22 are located on the spore surface and are synthesized in the mother cell cytoplasm.
- A proteolytic factor is released during late sporulation, but mature spores and forespores are protected.
- A galactosamine-6-phosphate polymer in revertant spores protects OL proteins from degradation.
Conclusions:
- The outermost layer of Bacillus megaterium spores is primarily composed of P48, P36, and P22 proteins.
- These proteins are deposited via non-disulfide bonds and are protected by a sugar polymer from proteolytic degradation after mother cell lysis.