30 Z. JOHN ORDAL 



On the basis of microbiological assay, paper chromatography, and column 

 chromatography information, together with negative results obtained in the 

 addition of specific amino acids or combinations of amino acids, we have 

 fairly conclusive evidence that the amino acids of the original medium, and 

 a- and y-amino butyric acids, and diamino pimelic acid do not specifically 

 enhance sporulation of PA 3679-h in 2%i trypticase broth. 



Inasmuch as 2% trypticase broth is virtually nonsporogenic, but mediums 

 containing 4% and over of trypticase, 2%) trypticase plus 2% acid digest of 

 casein, or even 2% trypticase plus 2% casein, yield spores if adequate thia- 

 mine is present, it is apparent that casein contains some unidentified com- 

 ponent which is required for sporulation. Growth in the higher concentra- 

 tions of casein derivatives yields larger cell crops than the 2% trypticase 

 with about 20% sporulation. When the spent 2% trypticase is refortified 

 with trypticase, the sporulation reaches as high as 90%. This may in part be 

 the result of the increased concentration of unidentified component of casein 

 which is required for sporulation. It does not explain, however, the high 

 sporulation rate obtained in the interrupted growth experiments, wherein 

 the second crop of cells taken from a medium yields up to 90% spores; 

 but if the original inoculum were allowed to grow and remain in the medi- 

 um indefinitelv, the cells would never produce many spores. Therefore, on 

 the basis of the information obtained from the studies in progress, we be- 

 lieve that during growth the organism synthesizes and elaborates into the 

 medium something which it requires for sporulation. 



Informal Discussion 



Harlyn Halvorson: Dr. Ordal has given us an excellent review of the 

 theories of sporulation. Inherent assumptions in all of these theories, es- 

 pecially the starvation theory defended by Grelet, involve the nature of 

 precursors of spore macromolecules. Irreversible commitment to sporula- 

 tion would involve either a redirection of de novo synthesis of macromole- 

 cules which are spore specific, or a modification of vegetative cell macro- 

 molecules prior to their incorporation into spores. Although Foster and 

 Perry have indicated the former applies in the case of some spore proteins, 

 it is not clear that this is a general phenomenon. A methodology for in- 

 vestigating de novo synthesis in the case of proteins and nucleic acids has 

 been forthcoming from the studies of Gale, Mandelstam, Cowie and our- 

 selves. Vegetative pools of amino acids, purines, pyrimidines and nucleo- 

 tides can be specifically suppressed with a resulting restriction in protein 

 synthesis, nucleic acid synthesis, or both. There are also an increasing 

 number of mutants available in the Bacillaceae to aid in specifically con- 

 trolling pool components. Also, by briefly exposing cells to C^^ labeled pre- 



