Trisporic Acid Biosynthesis in Blakeslea trispora via Mating Type-specific Precursors*

Abstract Separate (+) and (-) mating type cultures of Blakeslea trispora synthesized: (a) labeled trisporic acid B and trisporic acid C when incubated with labeled, partially purified extracts isolated from opposite mating type cultures; (b) unlabeled trisporic acids when incubated with labeled glucose and unlabeled extracts isolated from opposite mating type cultures; and (c) over 100-fold less trisporic acids when incubated with labeled extracts isolated from the same mating type cultures. Thus, separate (+) and (-) cultures synthesize mating type-specific precursors of trisporic acids.

SUMMARY Separate (+) and (-) mating type cultures of Blakedea trispora synthesized: (a) labeled trisporic acid B and trisporic acid C when incubated with labeled, partially purified extracts isolated from opposite mating type cultures; (5) unlabeled trisporic acids when incubated with labeled glucose and unlabeled extracts isolated from opposite mating type cultures; and (c) over lOO-fold less trisporic acids when incubated with labeled extracts isolated from the same mating type cultures.
Sutter and co-workers (4) demonstrated that separate (+) and (-) cultures of B. trispora synthesized small amounts of trisporic acid B and trisporic acid C when stimulated by a neutral fraction isolated from the culture medium of the opposite mating type culture of B. trispora.
In this paper, we demonstrate that the neutral fractions do contain mating type-specific precursors of trisporic acid B and trisporic acid C.
Labeled PNFi was prepared as follows. Ten 2-liter flasks, each containing 500 ml of PGT medium, were inoculated with (+) mycelia and incubated on a gyratory shaker at 25" (4). After 44 hours, 25 PCi of [ U-i4C]glucose (118 nmoles) were added to each flask and the incubation was continued for an additional 76 hours. The culture medium was collected and extracted in * This work was supported in part by National Science Foundation Grant GB-20533 from the Metabolic Biology Program.
$ Present address, Department of Pharmacology,-University of Witwatersrand.
(The culture medium was not adjusted to pH 2 prior to extraction, a step performed in previous work (4). This new procedure resulted in a 90 y0 rather than a 40 o/0 recovery of the trisporic acid-stimulating components from (+) culture medium.) The chloroform extract was evaporated to dryness in DUCUO, and the residue dissolved in 6 ml of ethanol and then purified by Sephadex LH-20 Ch romatography (4). The active fractions from the column contained 950,000 cpm and 2,550 A285 units of PNF.
Unlabeled I'NF containing 2,790 A285 units was prepared at the same time. I'NF absorbs ultraviolet radiation maximally at 285 nm. AzB5 units arc the A285 reading of the sample times the dilution factor times the total milliliters of undiluted sample solution.
Radioactivity measurements were made in a Beckman LS-230 liquid scintillation system after the sample solvent (ethanol) was evaporated from the counting vial by bubbling with nitrogen gas and after 5 ml of Bray's (7) solution without 1,4-bis[2-(5-phenyloxazolyllbenzene was added. Labeled PNF was added to a 36-hour (-) culture, (+) culture, and a 0.5-liter flask containing 100 ml of uninoculated PGT medium and then incubated for 2.5 hours. After the incubation, the culture media were collected, and the acid fractions were isolated and partially purified by DEAE-Sephadex chromatography (4). Column fractions, in which trisporic acids were eluted if present, were pooled and analyzed for radioactivity and A 325. (Trisporic acids absorb ultraviolet radiation maximally at 325 nm.) The purified acid fraction from the (-) culture, but not the (+) culture, contained both radioactivity (29,280 cpm) and ultraviolet-absorbing material (116 A 325 units) above the levels found in the purified acid fraction from uninoculated PGT medium (Table I). In a control experiment in which unlabeled PNF and labeled glucose had been incubated for 2.5 hours with a ( -) culture, (+) culture, and a flask containing uninoculated PGT medium, the purified acid fractions all exhibited the same low radioactivity (Table I). However, the purified acid fraction from the (-) culture, but not (+) culture, contained ultraviolet-absorbing material (122 Aa units) above the level found in the purified acid fraction from uninoculated PGT medium.
Trisporic acid B and trisporic acid C in purified acid fractions were resolved and purified by silica gel thin-layer chromatography (4). Trisporic acid B and trisporic acid C, isolated from the (-) culture incubated with labeled PNF, exhibited specific activities of 250 cpm per A325 unit.
These observations demonstrate that both trisporic acid C and trisporic acid B were radioactive and therefore PNF contains mating type-specific precursors of trisporic acids. Radioactive trisporic acid C and trisporic acid B were also isolated from uninoculated PGT medium incubated with labeled PNF, indicating that the PNF preparations contained the trace amounts of trisporic acids which accumulate in the culture medium of 5-day (+) cultures (4).
Similar experiments were performed with MNF. The quantity of trisporic acid-stimulating components synthesized by (-) cultures, however, is less than fsO the amount synthesized by (+) cultures (4). Werkman and van den Ende (6) reported that trisporic acids, isolated from (+ / -) culture medium by organic solvent extraction and partially purified by DEAE-Sephadex chromatography, stimulated synthesis of these components in (-) cultures of B. trispora. Therefore, labeled MNF was prepared as follows. Forty flasks, each containing 500 ml of PGT medium, were inoculated with (-) mycelia and incubated. Labeled MNF (320 ASO units) was added to each of five 34hour (+) cultures, (-) cultures, and flasks containing 100 ml of uninoculated PGT medium and then incubated for 5 hours. The purified acid fraction isolated from (+) cultures, but not (-) cultures, contained significant radioactivity (18,700 cpm) and ultraviolet-absorbing material (84 A325 units) above the levels found in the purified acid fraction from uninoculated PGT medium (Table II).
In a control experiment in which unlabeled MNF and labeled glucose had been incubated with (+) cultures, (-) cultures, and flasks containing uninoculated PGT medium, the purified acid fractions all exhibited the same low radioactivity (Table II).
However, the purified acid fraction from (+) cultures, but not (-) cultures, contained ultraviolet-absorbing material (91 AS25 units) above the level found in the purified acid fraction from uninoculated PGT medium.
Trisporic acid B and trisporic acid C, isolated from the purified acid fraction of (+) cultures incubated with labeled MNF, represented 65y0 of the A 325 units recovered from the silica gel chromat.ogram.
The remaining 35% was distributed between two unidentified compounds which migrated below trisporic acid C. Each trisporic acid exhibited a specific activity of 180 cpm per A325 unit. When the labeled trisporic acid C (11 As25 units) and authentic unlabeled trisporic acid C (99 A 325 units) were co-chromatographed on a Sephadex LH-20 column, the radioactivity and A325 were coincident.
A similar coincidence was observed when labeled trisporic acid B (3 A325 units) and authentic unlabeled trisporic acid B (27 A 325 units) were co-chromatographed. These observations demonstrate that both trisporic acid C and trisporic acid B were radioactive and therefore MNF contains mating type-specific precursors of trisporic acids.
The experiments reported in this communication differ from the tracer studies of Werkman and van den Ende (6) in three ways. (a) The final radioactivity measurements were made with pure trisporic acid C and trisporic acid B rather than with a partially purified extract of trisporic acids. (b) The radioactivity in the neutral fractions was shown to be incorporated into trisporic acids in a mating type-specific fashion.
(c) The labeled compounds in a neutral fraction were shown not to be degraded to metabolites of glucose prior to incorporation into trisporic acids.