22 Z. JOHN ORDAL 



factive Anaerobe 3679 sporulates in the presence of glucose; but in such 

 cultures the spores germinate, and spore yields are low unless they are har- 

 vested at exactly the right time. Similar results were also observed by Hal- 

 vorson (1956) and his co-workers. Hardwick and Foster (1952), using re- 

 placement culture techniques, presented rather convincing evidence that glu- 

 cose affects sporulation of B. mycoides. The vegetative cells were produced 

 in a glucose-glutamate salts medium; they were washed and resuspended in 

 replacement medium in which the glucose effect was evaluated. The adverse 

 effect of acidity resulting from glucose metabolism was overcome by using 

 phosphate buffer as the replacement solution. When the glucose was added 

 after five hours or less of shaking, sporulation was completely suppressed. 

 Further delay in the addition of glucose progressively reduced the inhibi- 

 tion. When glucose was added after 9 or 10 hours, full sporulation took 

 place. They concluded that the inhibitory effect of glucose was due to the 

 fact that glucose metabolism successfully competed with the intracellular 

 metabolism essential for sporogenesis. The early addition of glucose pre- 

 empted the use of nitrogenous precursors for sporogenesis whereas delayed 

 addition allowed sporogenesis to proceed to an irreversible stage. They sub- 

 stantiated the above assumption by demonstrating that the simultaneous 

 addition of airunonia counteracted the glucose-inhibitory effect. 



(b) Nitrogen sources. Hardwick and Foster (1952) have demonstrated 

 that cells of B. mycoides produced in a medium that is low in nitrogenous 

 material fail to sporulate when they are shaken in distilled water, whereas 

 cells produced in a nitrogen-rich medium sporulate readily. They concluded 

 that a cell impoverished in regard to its protein content loses its ability to 

 sporulate. They considered these results as additional evidence that spore 

 proteins are synthesized from cellular nitrogenous materials. 



Other investigations have been directed toward the specific effect of var- 

 ious amino acids. Williams and Harper (1951) show that the omission of 

 leucine from their chemically defined media reduced sporulation by B. 

 cereus. Krask (1953) reported that methionine sulfoxide, an antagonist for 

 the conversion of glutamic acid to glutamine, inhibited sporulation by B. 

 subtilis in concentrations which did not appreciably affect the vegetative 

 growth. He also cites unpublished data which indicated that more glutamic 

 acid was required for maximum sporulation than for growth. Blair (1950) 

 noted that the omission of methionine from a synthetic medium suppressed 

 sporulation of Clostridium botulinum. In our studies with B. coagulans, 

 we obtained the results presented in Table I. When the sulfur-containing 

 amino acids (methionine and cystine) were left out of the medium, sporu- 

 lation was markedly reduced. We thought, at that time, that we had dem- 

 onstrated a sporulation requirement for methionine, as the amount of growth 



