THE PRESENCE OF LOW-MOLECULAR-WEIGHT GERMINATIVE SUBSTANCES IN BACILLUS-CEREUS T-SPORE EXTRACT

An extract from intact spores of Bacillus cereus T having a germinatio n-inducing activity was studied. Two distinct germinative principles w ere found through dialysis of the extract. One was diffusible through the dialysis membrane and the other was non-diffusible. The activity o f the former fraction was inhibited by the addition of 1 mM glyeolethe rdiamine-N,N,N',N'-tetraacetic acid (GEDTA), whereas the latter fracti on was inactive unless GEDTA was added to the assay system. The diffus ible principle maintained the major portion of the activity found in t he crude spore extract. By means of high-performance liquid chromatogr aphy (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction. Of those fra ctions, two fractions (No. 1 and No. 8) were responsible for the germi nation-inducing activity, but no reconstituted activity was observed u nless both fractions No. 1 and No. 8 were added to the assay system. A mino acid analysis of fraction No. 1 revealed that the fraction was ri ch in free amino acids, especially in alanine. On the other hand, by t he use of reverse-phase HPLC and fast atom bombardment mass spectromet ry, it was concluded that the effective substance in fraction No. 8 wa s inosine. Based on these findings, it was suggested that the active s ubstances in fraction No. I might be a free amino acid such as L-alani ne and/or Ca2+ and a Ca2+-binding substance.