Heterothallism of mucoraceous fungi: A review of biological implications and uses in biotechnology

The phenomenon of heterothallism in filamentous fungi is reviewed, with emphasis on the discussion of hormonal regulation of heterothallic strains of mucoraceous fungi. This process is viewed from the standpoint of current understanding that fungal cells communicate with each other using a special “language,” i.e., signaling chemicals (hormones or pheromones). Physiological and biochemical criteria of distinguishing between heterosexual strains, which make it possible to draw analogies with higher eukaryotes, are set forth for the first time, based on experimental data obtained with Blakeslea trispora. The synthetic pathway to trisporic acids (a zygogenic sex hormone of Mucorales), their relation to carotenoids, and biological functions are described. The similarity (both structural and functional) between fungal, plant, and animal hormones is another topic dealt with. Current understanding of the role of terpenoids in the evolution of sexual communication and transduction is presented with an excursion into microbial endocrinology, a novel field of research in biology. The concluding part of the review analyzes the data on biotechnological implications of the phenomenon of heterothallism. Specifically, it may be used to obtain a series of isoprenoid compounds, such as β-carotene and lycopene (which exhibit pronounced antioxidant activity), as well as sterols and trisporic acids.     

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.     

Trisporoids under the stimulation of carotenogenesis in Blakeslea trispora

The patterns of trisporoid synthesis in joint cultivation of Blakeslea trispora mates have been studied. The pair of the not-zygospore-forming carotenoid overproducer strains T(+) and T(?) was found to synthesize a large amount of trisporoids, which did not differ in biological activity from those in the wild type strains. While the ??-carotene synthesis stimulator ??-ionone increased the amount of trisporoids, the share of trisporic acids in their composition decreased considerably. The lycopene synthesis stimulator 2-amino-6-methylpyridine caused a decrease in the content of trisporoid which had no trisporic acids in their composition. Emergence of a new substance with the maximum absorption at 250 nm, which accounted for up to 45% of the sum of trisporoids, was a general regularity in the action of both stimulators. The combined action of these two effectors resulted in additional stimulation of lycopene synthesis and was accompanied by the disappearance of trisporic acids. The aggregate findings indicate that both carotenogenesis stimulators inhibit the synthesis of trisporic acids, i.e., their action is not mediated by stimulation of trisporoid synthesis.     

Trisporoids and carotenogenesis in Blakeslea trispora

The recent data on the pathways of carotenoid biosynthesis, which resulted in revision of the earlier concepts, are analyzed. Trisporoid diversity and their role in the sexual process of mucoraceous fungi, resulting in formation of sexual cells (zygospores) are discussed. Special attention is paid to the role of trisporic acids in carotenogenesis of combined culture of the (+) and (?) strains, in which zygospores are not formed.     

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.     

Specificity and transformations of the trisporic acid series of fungal sex hormones

Chromatographic and spectroscopic characterizations and comparative bioassay data are given for trisporic acid A, the separate 9-cis- and 9-trans-isomers of trisporic acids B and C, and trisporol C, all obtained from 'mated' (plus and minus) cultures of Blakeslea trispora. All five acids show comparable levels of hormone activity on both the mating types of Mucor mucedo, whereas natural trisporol C more specifically affects a plus strain and the laboratory-derived methyl esters are minus-specific. Similarly plus and minus strains of B. trispora convert trisporol C and the esters into trisporic acids at different rates, and they effect different transformations of administered methyl ¹⁴C-trisporate C.     

Localization and partial characterization of a sex-specific enzyme in homothallic and heterothallic mucorales

A study was made of some late reactions in the trisporic acid biosynthetic pathway in Mucor mucedo. Trisporic acids induce sexual reproduction in several Mucorales.Two enzymes involved in these reactions, a NADP-dependent dehydrogenase and an esterase, appeared to be highly specific for the minus mating type.The synthesis of these enzymes is stimulated by trisporic acids, indicating a positive control of these hormones upon their own synthesis.The dehydrogenase was histochemically shown to be concentrated in the zygophores of Mucor mucedominus. In the homothallic Zygorhynchus moelleri the copulating main branch (which is known to have a minus character) appeared to be the major site of dehydrogenase activity.     

Aspergillus genomes: secret sex and the secrets of sex

The genomic sequences of three species of Aspergillus, including the model organism A. nidulans (which is homothallic: having no differentiated mating types, a strain being able to cross with itself), suggest that A. fumigatus and A. oryzae, considered to be asexual, might in fact be heterothallic (having two differentiated mating types, a strain being able to cross only with strains of opposite mating type). The genomic data have implications for the understanding of the evolution and the mechanism of sexual reproduction in this genus. We propose a model of epigenetic heterothallism to account for the reproductive patterns observed in Aspergillus nidulans.     

Sexual differentiation in Mucor: Trisporic acid response mutants and mutants blocked in zygospore development

Spores of a minus strain of Mucor mucedo (Bref.) were treated with 1-methyl-[3-nitro]-1-nitro-soguanidine and mutants were isolated either by testing for zygophore induction with externally supplied trisporic acids (TA) or by mating with wild type plus colonies. Mutants were found defective (Tar^?) or temperature-sensitive (Tar-Ts) in their reaction towards trisporic acids, blocked or temperature-sensitive in their mating with plus strain (Mat^? or Mat-Ts) or temperature-sensitive in zygospore development (Zyg-Ts). The inability to react against externally supplied trisporic acids was not necessarily coupled with an inability to mate with plus strain (phenotype Tar^? Mat⁺). This indicated that the diffusion and uptake of trisporic acids is not a necessary prerequisite to the sexual interaction of Mucor mating types.     

Relationship Between Sexuality and Carotene Synthesis in Blakeslea trispora

When stimulated by equivalent amounts of progametangia-inducing hormones, cultures of the minus mating type of Blakeslea trispora produce about the same quantities of carotenoids as mated cultures of the fungus, which suggests that the stimulation of carotene synthesis during the sexual activity of mated cultures is the result of hormonal action. These hormones were isolated and purified. From spectroscopic analysis of purified samples, it appears that the hormones are identical with trisporic acids B and C. When both mating types of B. trispora were cultivated in one vessel but were kept apart by membrane filters, the formation of sex hormones was not inhibited. Physical contact between the mating types is obviously not required for the induction of sexual activity. The sex hormones also formed in combined cultures of B. trispora-plus and Zygorhynchus moelleri (a homothallic species), but not in combined cultures of B. trispora-minus and Z. moelleri. This is evidence for the hypothesis that the hormones are produced by B. trispora-plus only.     

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.     

The correlation between the synthesis of β-carotene and zygote formation by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> heterothallic strains

It was demonstrated for the first time that the level of carotenogenesis by the heterothallic Blakeslea trispora strains intensively forming zygospores decreased under conditions of a surface cocultivation during their sexual interaction as compared with the strains grown separately. On the contrary, carotenogenesis was stimulated during a sexual interaction of the strains incapable of forming zygotes. In a submerged culture, the zygote-forming pairs of strains synthesized a considerably larger amount of trisporic acids but a smaller amount of carotenoids than the strains not forming zygospores. The discovered inverse dependence between zygote formation and carotenogenesis allowed us to suggest the inability to form zygotes as a criterion for selecting carotenogenic strain pairs.     

真菌有性生殖调控与进化

以真菌为对象的有性生殖机制研究揭示了普遍存在于真核生物中的生物学现象及规律,包括染色体倍性变化、减数分裂形成配子、交配对象识别及细胞一细胞融合形成合子等．真菌的有性生殖由交配型位点控制,除了类似其他真核生物两性生殖的异宗配合外,还包括同宗配合和次级同宗配合,部分物种的单倍体还具有交配型互换的能力．互补交配型的单倍体通过荷尔蒙及其受体进行相互识别,再经过G蛋白偶联受体介导的信号途径调控有性生殖过程及子实体发育,这一过程受多种胞内调控因子及外界环境条件的影响．不同真菌类群生殖方式的演化与物种进化仍缺少统一的规律．进一步研究揭示,真菌有性生殖的调控机制及环境诱导因子,不仅具有重要的理论意义,也有利于促进不同经济真菌子实体的人工培养及高效利用．     

Unidirectional evolutionary transitions in fungal mating systems and the role of transposable elements

In the fungal kingdom, the evolution of mating systems is highly dynamic, varying even among closely related species. Rearrangements in the mating-type (mat) locus, which contains the major regulators of sexual development, are expected to underlie the transitions between self-sterility (heterothallism) and self-fertility (homothallism). However, both the genetic mechanisms and the direction of evolutionary transitions in fungal mating systems are under debate. Here, we present new sequences of the mat locus of four homothallic and one heterothallic species of the model genus Neurospora (Ascomycota). By examining the patterns of synteny among these sequences and previously published data, we show that the locus is conserved among heterothallic species belonging to distinct phylogenetic clades, while different gene arrangements characterize the four homothallic species. These results allowed us to ascertain a heterothallic ancestor for the genus, confirming the prediction of the dead-end theory on unidirectional transitions toward selfing. We show that at least four shifts from heterothallism to homothallism have occurred in Neurospora, three of which involve the acquisition of sequences of both mating types into the same haploid genome. We present evidence for two genetic mechanisms allowing these shifts: translocation and unequal crossover. Finally, we identified two novel retrotransposons and suggest that these have played a major role in mating-system transitions, by facilitating multiple rearrangements of the mat locus.     

大球盖菇性遗传模式和育种工艺研究

为探讨大球盖菇的交配型,以鲜大球盖菇为材料,经担孢子弹射,稀释分离法获得22个单孢菌株。经单孢出菇验证其性遗传模式为异宗结合。再经单孢配对试验,得到4种不同交配型的单孢菌株,单孢的极性比为4∶4∶11∶3。经异核体菌株出菇试验,证实大球盖菇的性遗传模式为四极性异宗结合。     

Sex specificity of hormone synthesis in Mucor mucedo

Sex specificity is observed in the mating types of the fungus Mucor mucedo with respect to the production of 4-hydroxy methyltrisporates (plus mating type) and trisporins (minus mating type), and in the conversion of these metabolites to trisporic acids by the mating partner. These compounds induce zygophores on the opposite mating type only.     

Chemical regulators of carotenogenesis by Blakeslea trispora

The activation of the carotene biosynthetic pathway in Blakeslea trispora was found to occur by trisporic acid and many other compounds such as abscisic acid, β-ionone, α-ionone and vitamin A which share significant structural similarity with trisporic acid. The magnitude of stimulatory activities of these effectors was in the order trisporic acid > abscisic acid > β-ionone > α-ionone > vitamin A. Comparison of structures and stimulatory activities of all the effectors indicated that the short length of the side chain and the presence of a keto group in the ring structure of the trisporic acid molecule contributed significantly to the biological activity towards carotenogenesis.     

Organic acids production by white rot Basidiomycetes in the presence of metallic oxides

The purpose of the present work was to determine if selected fungal strains belonging to wood-rotting Basidiomycetes are able to grow on and to solubilize different insoluble oxides in solid media. Twenty-eight strains of white rot fungi were checked for their growth on oxide-amended media (ZnO, CaO, Cu2O). All strains displayed growth on Zn-amended plates but to a different extent, and Cu2O-amended plates turned out to be the most toxic oxide. Most of the tested strains solubilized oxalates and produced noticeable clear zones under the mycelium. These clear zones were tested for the presence of organic acids, the level of which was clearly elevated upon exposure of fungal strains to insoluble oxides. We determined the presence of oxalic, malic, and formic acids, with oxalic acid the predominant one.     

不同碳源对三种溶磷真菌溶解磷矿粉能力的影响

通过液体培养法 ,对 3种溶磷真菌利用葡萄糖、果糖、蔗糖、麦芽糖、淀粉和纤维素等碳源溶解宜昌产磷矿粉的试验 ,结果表明 ,菌株P2 3在供给葡萄糖时的溶磷能力最高 ,并在一定程度上能够利用长链碳源淀粉和纤维素为营养而溶磷 ;而高效溶磷菌株P6 6和P39溶磷的最佳碳源是果糖和麦芽糖 ,该两菌株利用淀粉和纤维素的溶磷效果很小 ,甚至不溶磷。 3种溶磷真菌培养滤液 pH值、可滴定酸含量与其溶磷量之间的相关性因菌株而异 ,差别很大。菌株P2 3培养滤液pH值、可滴定酸含量与其溶磷量之间相关性很低 ,但菌株P6 6和P39培养滤液pH值、可滴定酸含量与其溶磷量之间相关性却达到极显著水平 (P <0 0 1)。结果表明 ,不同碳源对溶磷菌溶解磷矿粉能力影响很大 ,分析推断 3种菌株产生的有机酸活化磷矿粉能力为P6 6>P39>P2 3。     

Sex determination in the first-described sexual fungus

The original report of sex in fungi dates 2 centuries ago to the species Syzygites megalocarpus (Mucoromycotina). The organism was subsequently used in 1904 to represent self-fertile homothallic species when the concepts of heterothallism and homothallism were developed for the fungal kingdom. In this study, two putative sex/MAT loci were identified in individual strains of S. megalocarpus, accounting for its homothallic behavior. The strains encode both of the high-mobility-group domain-containing proteins, SexM and SexP, flanked by RNA helicase and glutathione oxidoreductase genes that are found adjacent to the mating-type loci in other Mucoromycotina species. The presence of pseudogenes and the arrangement of genes suggest that the origin of homothallism in this species is from a heterothallic relative, obtained via a chromosomal rearrangement to switch two alleles into two separated loci within a single genetic background. Similar events have given rise to homothallic species from heterothallic species in ascomycete fungi, demonstrating that conserved forces shape the evolution of sex determination and speciation in highly diverged fungi.