In Clostridium perfringens S40, spore germination-specific enzymes are synthesized during sporulation. Previous reports have demonstrated that two cortex-lytic enzymes, SleC and SleM, and a component of germination-specific protease, CspC, are located outside the cortex as an integral part of the dormant spore. In the present study, we examined the time and compartment of these enzymes' gene expression using reverse transcription-PCR (RT-PCR) and fluorescence microscopy on green fluorescence protein (GFP)-fused proteins. These results suggested that CspABC, SleC, and SleM are synthesized in the mother cell compartment of sporulating cells, probably at stages II approximately III of sporulation, and that the expression of cspABC genes is tricistronic.

SummaryWe found that dehydrated legume seeds (6 genera, 19 species and cultivated varieties) contained considerable amounts of vitamin C (VC). The average value of total VC content per 100 g of dry weight in dehydrated seeds varied from 0.24 mg (kidney beans) to 4.14 mg (green peas). Yard beans showed highest values among all legumes examined here in the both dehydrated and rehydrated forms (3.19 and 10.8 mg, respectively). By soaking for 16 h in the dark at 20˚C, total VC contents of black grams and mung beans increased to 3.1-and 4.5-fold, respectively. However, three varieties of green peas (Hakuryu, Kurumeyutaka, and Nankaimidori) significantly lost their VC during the same soaking treatment. Total VC content of a rehydrated and cooked mung beans was higher than that of a dehydrated form. Appreciable amounts of total VC were detected in the immature seeds of six different genera such as yard beans, kidney beans , broad beans , green peas, soybeans and peanuts. Except for mung beans, 70-100% of VC in dehydrated seeds of adzuki beans, broad beans, green peas, black soybeans, and soybeans was lost by boiling. Total VC and L -ascorbic acid in mung beans, green peas, broad beans, black soybeans, and adzuki beans remained even after boiling, suggesting that it is possible to obtain VC from the cooked forms of these legume seeds.

The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB 1-108 -GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB 1-108 -GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic δ -lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB 1-45 -GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic δ -lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the δ -lactam structure of the cortex, not the formation of normal coat layer.

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