Mechanism of the molecular construction of germination apparatus of bacterial spores through self-assembly and subcellular localization of germiantion-related enzymes.

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15580060
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied microbiology
• Research Institution: NAGOYA UNIVERSITY
• Principal Investigator: MORIYAMA Ryuichi Nagoya University, Graduate School of Bioagricultual Sciences, Associate Professor, 大学院・生命農学研究科, 助教授 (60191061)
• Project Period (FY): 2003 – 2004
• Keywords: bacterial spores / cortex-lytic enzyme(s) / germination / sporulation / protein-protein interaction / enzyme activation / protease

Research Abstract

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.