Trisporic acid synthesis in Blakeslea trispora.

Cultures of the (-) strain of Blakeslea trispora synthesize trisporic acid C, a sex hormone, from precursors made by cultures of the (+) strain. The precursors are nonacidic compounds isolated from the culture medium of the (+) strain by chloroform extraction. Actidione does not inhibit trisporic acid synthesis from isolated precursors, suggesting that the enzymes involved in the synthesis in cultures of the (-) strain are constitutive.

Mutations affecting sexual development in Phycomyces blakesleeanus.

Although zygospore (mature zygote) formation in P. blakeslleeanus occurs in liquid glucoseglutamate medium, morphological observations are made more easily when cultures are grown on 1-mm-thick agar medium. Zygophores (sexually differentiated hyphae) develop prior to physical contact in crosses of (plus) and (minus) wild types. Zygophores interlock upon contact and then undergo six successive morphological changes to become a zygospore. Mutants with abnormal carotene synthesis exhibit aberrant sexual behavior. Some zygospores do form in crosses of carA mutants and wild types. Only paired zygophores form in crosses of wild type(plus) with car-42(minus), a beta-carotene-accumulating mutant. Zygophores form only on (minus) in crosses of wild type(plus) with carB(minus), carR(minus), carAcarR(minus), and carBcarR(minus) mutants, and only on (plus) in crosses of car-43(plus) with wild type(minus), car-42(minus), and carA(minus) mutants. Zygophores do not form in crosses of car-43(plus) with carB(minus), carR(minus), carAcarR(minus), and carBcarR(minus) mutants. These observations demonstrate that each mating type makes a chemical messenger that stimulates zygophore development in the opposite mating type.

Comparison of stainless-steel closures and cotton plugs in maintaining contamination-free medium in circulating air.

Culture flasks capped with stainless-steel closures became contaminated when incubated in circulating air. Cotton plugs prevented this contamination.

Separation of beta-factor synthesis from stimulated beta-carotene synthesis in mated cultures of Blakeslea trispora.

Mated cultures of Blakeslea trispora, grown in a potato extract-glucose-thiamine medium, produced 10 to 15 times more beta-carotene than either unmated culture. Mated, but not unmated, cultures produced a family of compounds (beta factor) which stimulated carotenogenesis in unmated cultures. In fact, carotenogenesis was stimulated sixfold more in minus cultures than in plus cultures. By altering the relative amounts of plus and minus inocula used in fermentations of mated cultures, it was possible to separate the synthesis of beta factor from the synthesis of extra beta-carotene. The plus strain appeared to produce the beta factor; the minus strain appeared to produce most of the extra beta-carotene. Kinetic studies of beta-factor formation suggested that physical contact between the two strains may be required to initiate beta-factor synthesis.

Methyl trisporate E. A sex pheromone in Phycomyces blakesleeanus.

Combined mating type cultures of Phycomyces blakesleeanus accumulate 41 mg of trisporic acids/l of medium, of which 30% is trisporic acid E. The methyl ester of trisporic acid E exhibits the same zygophore -inducing activity in bioassays with P. blakesleeanus and Mucor mucedo as does the pheromone methyl trisporate C. The structure of methyl trisporate E is 1,5-dimethyl-2-hydroxyl-4-oxo-6-(2'-hydroxyl-6'- methylocta -5',7'-d ien-8'-yl) -5-cyclohexene-1-carboxylic acid methyl ester.

Trisporic acid biosynthesis in separate plus and minus cultures of Blakeslea trispora: identification by Mucor assay of two mating-type-specific components.

Separate plus and minus cultures of Blakeslea trispora synthesize small amounts of trisporic acids under specific conditions. These amounts are expressed as a percentage of the trisporic acids (50 mg/liter of medium) synthesized by mixed plus-minus cultures in 5 days. Plus cultures, without additives from minus cultures, synthesize 0.1% trisporic acids. Plus cultures synthesize 0.4% trisporic acids when stimulated by M-factor, a mating-type-specific component synthesized by minus cultures. Minus cultures, without additives from plus cultures, do not synthesize even 0.0001% trisporic acids. Minus cultures synthesize 1% trisporic acids when stimulated by P-factor, a mating-type-specific component synthesized by plus cultures. Minus cultures synthesize M-factor when stimulated by pi, a component synthesized by plus cultures. We speculate that (i) minus cultures synthesize a component, mu, which stimulates P-factor synthesis in plus cultures, and (ii) both M-factor and P-factor are precursors of trisporic acids.

(-) mating type-specific mutants of Phycomyces defective in sex pheromone biosynthesis.

We have isolated the first mating type-specific mutants in mucoraceous fungi. Both mutants in Phycomyces blakesleeanus appear to be defective in the same gene. The gene, present in both mating types, is necessary only in cultures of the (-) mating type. The gene codes for an enzyme in sex pheromone biosynthesis. The pheromone precursor made by the mutants is detectable only in cross-feeding experiments. The biological and solubility properties of the precursor suggest the precursor is 4-dihydrotrisporin, a metabolite of beta-carotene. Separate studies with beta-carotene-deficient mutants and Compound-P, a new chemically synthesized precursor of the pheromones, imply the constitutive level of enzymes for pheromone biosynthesis in Phycomyces is extremely low. In comparison, the level of enzymes for pheromone conversion to trisporic acid is higher. The mating type-specific mutants also catalyze the conversion of (+) pheromone to trisporic acid. This finding was unexpected because literature models predicted this reaction was catalyzed by the same enzyme which catalyzed the conversion of 4-dihydrotrisporin to (-) pheromone-a reaction missing in the (-) mating type-specific mutants. Thus, we propose a revised model for trisporic acid biosynthesis.

Cultures of separated mating types of Blakeslea trispora make D and E forms of trisporic acids.

Trisporic acids are end products of the sex-specific pheromones in mucoraceous fungi. We have found three new trisporic acids in cultures of Blakeslea trispora in which (+) and (-) mating types were separated by a membrane with 0.45-microns pores. Two of the trisporic acids were new compounds; the structure of the third [previously described by Miller and Sutter [(1984) J. Biol. Chem. 259, 6420] as methyl trisporate-E with a hydroxyl group at C-2] was revised. Trisporic acid-E(3R), trisporic acid-E(3S), and trisporic acid-D(2S) were in a 1:1:2 ratio, accounted for 9% of the total trisporic acids, and differed by the position and configuration of a hydroxyl group on the ring at C-2 or C-3, the conformation of the ring, the extent of rotation of the side chain relative to the ring, and either a carbonyl or hydroxyl group on the side chain at C-13. These three compounds accounted for only 0.5% of the total trisporic acids in combined mating type cultures. Since the combined cultures did not metabolize trisporic acid-E(3R), its biosynthesis apparently ceases when opposing mating types contact each other physically. We speculate that B. trispora and Phycomyces blakesleeanus utilize different pheromones to regulate an early event (possibly zygotropism) in sexual development.