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The levels of cAMP and cGMP were determined throughout the mitotic cycle of two independent strains of the naturally synchronous slime mold, Physarum polycephalum. The normal range of values was approx. 0.5–2.5 pmoles cAMP/ mg protein and 0.01–0.08 pmoles cGMP/mg protein. In our standard laboratory strain, there was no systematic pattern to the variations in the values within the normal range and no unique time in the cycle when values significantly above normal were noted. In the other strain recently cultured from spherules, elevated levels of cGMP, but not cAMP, were observed in late G2 and in the S phase. The data suggest that elevated levels of these cyclic nucleotides are not required for normal progression of the Physarum mitotic cycle which is unperturbed by artificial synchronizing procedures.

Daniel, John W. (University of Wisconsin, Madison, Wis.) and Harold P. Rusch . Niacin requirement for sporulation of Physarum polycephalum . J. Bacteriol. 83: 1244–1250. 1962.—The myxomycete Physarum polycephalum undergoes sexual sporulation if exposed to light after 4 days of incubation in the dark on a salts medium containing niacin, niacinamide, or tryptophan. None of these compounds is required for growth. Quinic acid, shikimic acid, intermediates of the kynurenine pathway, diphosphopyridine nucleotide (DPN), and triphosphopyridine nucleotide (TPN) replace niacin but a number of other tryptophan metabolites do not. Analogues of niacin inhibit sporulation when added at the beginning but not at the end of dark incubation with niacin. Folic acid, p -aminobenzoic acid, and p -aminobenzenesulfonamide inhibit sporulation if added at any time during the incubation or illumination periods. Reduced di- or triphosphopyridine nucleotide, but not DPN or TPN, reverse the p -aminobenzoic acid inhibition but do not replace the light requirement or shorten the dark incubation period. Gluconate and 2-ketogluconate also replace niacin. Glucose, pyruvate, malate, and oxalacetate inhibit the niacin-induced sporulation. Iodoacetate and fluoride do not counteract the glucose effect or inhibit sporulation.

Daniel, John W. (University of Wisconsin, Madison), Jacqueline Kelley, and Harold P. Rusch. Hematin-requiring plasmodial myxomycete. J. Bacteriol. 84:1104–1110. 1962.—The myxomycete Physarum polycephalum, previously shown to require chick embryo extract for growth on a partially defined, soluble medium, grows as well if hematin or certain hemoproteins are substituted for the embryo extract. Hematin is also required as a growth factor if the organism is grown on a synthetic medium. Of the variety of porphyrins tested only iron protoporphyrin IX is utilized for growth by P. polycephalum. Protoporphyrin IX is inactive. Protein-bound iron porphyrin is active at one-tenth the concentration of free hematin. Although hematin completely replaces embryo extract, the extract activity has properties not characteristic of hematin or the hemoproteins tested: ladility to light and rapid plasmodial uptake.

Daniel, John W. (University of Wisconsin, Madison), and Karlee Babcock . Methionine metabolism of the myxomycete Physarum polycephalum . J. Bacteriol. 92: 1028–1035. 1966.—Previous studies have shown that Physarum polycephalum requires exogenous methionine for growth, but not cysteine, folic acid, or vitamin B 12 . Methionine can also serve as the sole source of sulfur for all cellular requirements, without limiting the growth rate. S -methyl- l -cysteine, 2-hydroxy-4-methiol butyric acid, S -adenosyl- l -methionine, and methionine peptides were the only compounds supporting growth, when substituted for methionine. Other methionine analogues, methyl donors in combination with homocysteine, and intermediates of the cystathionine pathway were not active. Ethionine and S -ethyl cysteine were good methionine antagonists. This myxomycete is apparently unable to synthesize the methyl or S -methyl group, although it still appears able to transmethylate, at least from S -methyl cysteine, and probably from S -adenosyl methionine, which can also serve as a source of adenine.

Daniel, John W. (University of Wisconsin, Madison), Karlee L. Babcock, Alice H. Sievert, and Harold P. Rusch . Organic requirements and synthetic media for growth of the myxomycete Physarum polycephalum . J. Bacteriol. 86: 324–331. 1963.—An isolate of Physarum polycephalum was maintained in submersed pure culture in a completely defined medium, giving cell yields approximately 70% of those obtained in a partially defined medium. Optimal concentrations of the absolute organic requirements, identified as d - or l -methionine, biotin, thiamine, and hematin as previously reported, were established. Glycine was highly stimulatory. In a minimal medium, alanine or glutamine, not required in the complete medium, stimulated growth and appeared to function as sources for transamination. Arginine was also required in the minimal medium. The cell yield in a simplified medium containing the absolute requirements plus glycine, alanine, and arginine was approximately the same as that in the complete medium, but the growth rate was about 25% lower.

Daniel, John W. (University of Wisconsin, Madison) and Harold P. Rusch . Method for inducing sporulation of pure cultures of the myxomycete Physarum polycephalum . J. Bacteriol. 83: 234–240. 1962.—Techniques for inducing sporulation of pure cultures of Physarum polycephalum are described. Small plasmodia grown in agitated culture were harvested, allowed to fuse into large plasmodia (1.3 g wet weight each), and, after incubation in the dark on a salts medium, exposed to light for 2 hr. The ensuing formation of sporangia containing spores was complete after 12 to 16 hr. The obligatory conditions for sporulation are: (i) an optimal growth age occurring just prior to the maximal growth of the organism and at a time when nutrients in the medium are exhausted; (ii) 4 days of incubation on a medium containing only inorganic salts and niacin or tryptophan; and (iii) subsequent illumination with light of wavelengths between 350 and 500 mμ.

The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2 mM) and Ro-20-1724 (18 microM) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by gamma radiation. These results (1) emphasize the organism and agent specificity of cyclic nucleotide responses, (2) indicate that the kinetics and apparent refractory response are compatible with the function of previously identified membrane receptor systems, i.e., a blue light receptor and a choleragen receptor mediating cyclic AMP responses, and (3) suggest a relationship between the agent-specific cyclic nucleotide responses and the modification of gamma-radiation-induced delay of mitosis by the same agents, i.e., a decrease in the delay by those which elevate cyclic GMP levels and an increase in the delay by those which elevate only cyclic AMP levels.