CHEMICAL CHANGES DURING SPORE GERMINATION 73 



10 to 15 percent of the total germination exudate. Its constituents were 

 ae-diaminopimelic acid (DAP I, alanine, glutamic acid, and hexosamines 

 occurring in the molecular ratio of 1:3:1:8. Non-dialyzable peptides of very 

 similar constitution were present in extracts from disintegrated resting spores 

 of Bacillus species (Strange and Powell, 1954 (. The remaining constituents 

 of the germination exudate have not yet been fully investigated; they appear 

 to be mainly peptides of lower molecular weight and of different composition 

 from the non-dialyzable fraction. 



The Origin of the Constituents of the Germination Exudate 



At the moment, we do not know how calcium dipicolinate is distributed 

 within the resting spore. An X-ray examination of crystalline calcium dipi- 

 colinate and of air-dried resting spores of B. megaterium was kindly carried 

 out for us by Dr. M. M. Bluhm and Dr. J. C. Kendrew of the Cavendish 

 Laboratory, Cambridge. They obtained a characteristic crystalline-powder 

 diagram with calcium dipicolinate, but could find no evidence of a crystalline 

 structure in resting spores. It seems likely that calcium dipicolinate is in- 

 corporated within the spore protoplasm through chelate linkages of calcium 

 with protein, and that this arrangement may contribute to the heat-resisting 

 properties of the intact resting spore. 



The non-dialyzable peptide containing DAP and hexosamines appears to 

 be derived from the spore coat (Strange and Dark, 1956). Spore coat prepa- 

 rations from B. megaterium obtained by mechanical disintegration still con- 

 tained considerable amounts, and those from B. subtilis smaller amounts of 

 bound hexosamine. This was slowly released in the form of the DAP- 

 hexosamine peptide when the spore coat preparations were incubated in 

 distilled water or buffer solutions. In B. cereus it appeared that the peptide 

 was readily and completely released during mechanical disintegration. 



Independent evidence for the location of the DAP-hexosamine peptide on 

 the spore surface has recently been obtained from electrophoretic studies 

 (Douglas, 1955). From a comparison of the electrophoretic behavior of 

 resting spores of B. megaterium and B. subtilis, it was concluded that these 

 spores had different surface compositions. It was tentatively suggested that 

 the surface layer of the B. subtilis spore contained polysaccharide, whereas 

 that of B. megaterium contained lipid possibly complexed with protein. In 

 order to provide "standards of reference" to assist in the interpretation of 

 results, the electrophoretic behavior of inert particles coated with various 

 typesiof protein, lipid, and polysaccharide was recently studied (Douglas and 

 Shaw, 1956). In this series was included a preparation of the DAP- 

 hexosamine peptide isolated from germination exudates of B. megaterium. 



