Regulation of the germination-specific cortex-lytic enzymes of bacterial endospores by protein-protein interaction during sporulation.

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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. Thus exploring the mechanisms of regulation of germination-specific cortex-lytic enzymes during during germination is important to understand the molecular process of germination. We obtained the following results during the research aided by this grant.1. YpeB is encoded in sleB-ypeB operon, and spore germination of ypeB-deficient Bacillus subtilis is incomplete like that of sleB mutant. We confirmed previously reported undetectability of SleB in ypeB mutant spores by biochemical subcellular fractionation. However, SleB-GFP fusion proteins were observed to be located in the outside of spores during sporulation in both wild-type and ypB-deficient strains. Furthermore, immunoelectron … More microscopic observations indicated that SleB is normally localized inside of coat layer of the mutant spores as in the case of wild-type. These results require further investigations to determine the function of YpeB, especially in relation to the mode of existence of SleB in dormant spores and the activation of SleB during spore germination.2. We also studied the activation process of a SleC, a spore cortex-lytic enzyme of Clostridium perfringens. We found that inactive proenzyme is converted to active enzyme by germination-specific protease GSP during germination. The protease was a cysteine-dependent serine protease and GSP is likely localized with SleC on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reaction. SleC was most likely a bifunctional enzyme possessing lytic transglycosylase and N-acetylmuramoyl-L-alanine amidase activities. These results suggest that the mechanism on spore cortex hydrolysis during germination is not conserved between Bacillus species and C. perfringens. Less