When spores were suspended in distilled water and meat extract agar was used as a medium for colony formation, sublethal condition for the spores of
B. subtiliswas heating at80°for 30 minutes.
What kinds of amino acids were released from spores suspended indistilled water by heating at various temperatures for 30 minutes, especially at sublethalconditions, were examined by paper electrophoresis and paper chromatography. It was shown that glutamic acid was released faintly at 60°C and 70°C and clearly at 80°C. Glutamic acid, aspartic acid, arginine, lysine and probably ornithine were released at 90°C, 100°C and 120°C. In all cases peptide appeared. By hydrolysis of this peptide it was shown that peptide consisted of glutamic acid, aspartic acid, arginine, lysine and neutral amino acids.
As glutamic acid was released from spores by heating at under sublethal and sublethal temperatures, effects of glutamic acid on the colony formation of the spores heated at sublethal conditions were examined in synthetic medium. It was clearlyshown that glutamic acid was necessary for the colony formation of the spores heated at sublethal conditions in addition to amino acid which stimulated spore germination. Arginine had also good effect on the colony formation of that spores except glutamic acid.
L-alanine, even if acted as a stimulative amino acid on spore germination, had an inhibitory effect on the colony formation with glutamic acid.
The selationship between the requirement of amino acids for colony formation of the heated spores and the condition of heating of spores was as follows:
a) When spores are heated at relatively low temperature, glutamic acid is not necessary for the colony formation.
b) When spores are heated at relatively high temperature, only glutamic acid (or arginine) supports colony formation with amino acid which stimulates spore germination.
c) When spores are heated at higher temperature, colony formation is impossible.
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