|Budget Amount *help
¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 2004 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 2003 : ¥3,000,000 (Direct Cost : ¥3,000,000)
The cortex, a thick layer of peptidoglycan specific to bacterial spores, is responsible for the maintenance of dormancy and heat resistance of spores. Cortex hydrolysis during germination induced by specific nutrient germinants like L-alanine leads to a rapid loss of dormancy and thermostability of spores. Germination is a process controlled by the sequential activation of a set of pre-existing germination-related enzymes (germination apparatus). Thus exploring the mechanisms of molecular construction of germination apparatus during sporulation is crucial to understand the molecular process of germination. We obtained the following results during the research aided by this grant.
1.YpeB is encoded as sleB-ypeB operon in Bacillus subtilis genome, and spore germination of ypeB-deficient B.subtilis is incomplete like that of sleB mutant, suggesting that YpeB protein may be involved in germination process of B.subtilis spores. We confirmed that sleB-ypeB operon is conserved in other Bacillus species. We found that, as in the case of B.subtilis spores, B.cereus YpeB is processed into 30 kDa fragment during germination and released into germination exudate. N-terminal sequence of 30 kDa fragment of B.cereus and Bacillus subtilis YpeB was virtually identical, suggesting that the processing site of YpeB during germination should be conserved among Bacillus species. The site-directed mutagenesis of B.subtilis YpeB affected to the spore germination of B.subtilis. Furthermore, we found the protease activity, which cleaves GST-YpeB fusion protein around the processing site indicated above, in germination exudate of B.cereus spores
2.We also found that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase and N-acetylmuramoyl-L-alanine amidase activities, suggesting that the mechanism on spore cortex hydrolysis during germination is not conserved between Bacillus species and C.perfringens.