Molecular Characterization of Laccase Genes from the Basidiomycete Coprinus cinereus and Heterologous Expression of the Laccase Lcc1

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K_m of 21 ± 2 μM and a catalytic constant of 200 ± 10 min⁻¹ for O₂ with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

Secretional expression of a Bacillus subtilis xylanase gene in the Basidiomycete Coprinus cinereus

The Bacillus subtilis endo (β-1,4-) D-xylanase structural gene (xyn) was trimmed away from its signal sequence and then fused after the signal sequence of the basidiomycete Pleurotus ostreatus manganese(II) peroxidase cDNA. The resulting modified gene (xyn′) was inserted between the promoter and terminator of two chromosome-integrating, heterologous protein expression vectors. These recombinant plasmids were introduced into protoplasts of the monokaryotic Coprinus cinereus trp1 strain with the C. cinereus TRP1-containing plasmid. One Trp⁺ Xyn⁺ transformant for each of the recombinant plasmids was obtained, which showed a markedly high xylan-degrading activity as compared with the control Trp⁺ transformant.     

担子菌交配型基因的克隆及功能研究进展

担子菌中已有２０多个交配型基因被克隆,Ａ、Ｂ位点的交配型基因具有明显不同的结构特征。Ａ位点的交配型基因编码两类含相似同源结构域的蛋白质,两种蛋白质形成异源二聚体,调节依赖Ａ因子的发育过程;而Ｂ位点的交配型基因编码信息素及其受体。交配型基因编码的同源结构域蛋白质在植物、动物和菌类中都具有广泛的保守性。     

Patterns of Mating and Mitochondrial DNA Inheritance in the Agaric Basidiomycete Coprinus Cinereus

Patterns of mating and mitochondrial DNA (mtDNA) inheritance were investigated for the Basidiomycete, Coprinus cinereus in order to better understand the relationship of reproductive biology and mtDNA evolution in fungi. Results showed that the unique mating system of basidiomycetes can lead to the formation of mitochondrial mosaics (i.e., colonies composed of sectors differing in mtDNA). Mitochondria do not migrate along with nuclei during mating. Intracellular mixed or recombinant mtDNA molecules were not observed. Interestingly, it was found that mating asymmetry, caused by nonreciprocal nuclear migration, may be an important part of the reproductive biology of C. cinereus.     

Mating Type Switching in the Tetrapolar Basidiomycete Agrocybe Aegerita

The study of fruiting in the basidiomycete Agrocybe aegerita has shown that some haploid homokaryotic strains can spontaneously switch their mating specificities at the two unlinked A and B mating type factors. This event causes the dikaryotisation of primary homokaryons without plasmogamy and leads to the differentiation of sporulating fruit-bodies (pseudo-homokaryotic fruiting). For each mating type factor, the genetic analyses have revealed that: (1) parental and switched mating types segregate meiotically as Mendelian markers, (2) a total of six switched mating type factors (two parental and four nonparental) were obtained from a wild strain, (3) most of the nonparental factors have specificities differing from those of a large series of wild factors, (4) strains with the same expressed mating type can generate different specificities, (5) switching is always restricted to the same mating type in a homokaryon, (6) nonparental types can switch again, and (7) meiosis fixes the specificities to which switching can occur. This suggests, for the first time in filamentous fungi, the existence of a mechanism analogous to the mating type switching in yeasts. We hypothese that both A and B mating type regions in A. aegerita are constituted of three loci, one specialized in expression and two other carrying silent information. Mating type switching in homokaryotic strains would occur by copy transposition of silent A and B information into the expression loci. Moreover, we propose that during meiosis the silent loci are substituted by copies of the expressed loci.     

蓝光作用下黑曲霉形态发育与消减文库的分析

以持续黑暗培养为对照,采用扫描电镜观察了蓝光对黑曲霉孢子发育的影响,结果表明蓝光处理使菌丝粗壮,孢囊增大。以黑暗培养至36h再经蓝光处理3~4h的菌丝为实验组,对应时间段的黑曲霉菌丝为对照组,采用抑制性消减杂交(SSH)方法,构建了蓝光作用下黑曲霉的差异表达基因的cDNA文库。阳性克隆菌落经PCR扩增出200~500bp差异表达基因的cDNA片段,对序列进行同源性分析后发现两个差异表达的未知基因片段,为进一步研究蓝光诱导下黑曲霉差异表达的未知基因的功能提供依据。差异表达的基因片段大都与黑曲霉线粒体氧化还原酶类同源;荧光定量PCR分析结果表明所选取的部分差异基因片段在蓝光诱导下表达量均有提高。     

Molecular Polymorphism in the MTA and MTB Mating Type Genes of Tetrahymena thermophila and Related Asexual Species

Each of the seven mating types of Tetrahymena thermophila is determined by a pair of large genes, MTA and MTB, whose expression peaks at early conjugation. Each protein consists of a mating‐type specific domain and a common transmembrane domain. To assess variation in natural populations, regions of both domains from wild isolates expressing mating types V and VII were analyzed. Corresponding regions of amicronucleates incapable of mating also were examined. MTA and MTB showed high haplotype diversity, with greater sequence variation in MTB. Mating type VII was less variable than mating type V, suggesting more recent origin. No polymorphism distinguished between mat1‐ and mat2‐like alleles encoding different arrays of mating types, nor did polymorphisms give evidence of population structure. MTA and MTB variants have different phylogenies, suggesting independent rather than concerted evolution, and are under weak purifying selection. Codon usage is less biased than for housekeeping genes, and reassigned glutamine encoding stop codons are preferentially used. Amicronucleate T. thermophila and closely related nsp15 and nsp25 have higher levels of nucleotide and amino acid substitution, consistent with cox1 distances. The results suggest that complete sequencing of mating type genes of wild isolates coupled with functional analysis will be informative.     

Inhibition of Coprinus cinereus by 5-fluoroindole

5-Fluoroindole (5FI) was more inhibitory than 5-fluorotryptophan (5FT) to Coprinus cinereus. A mutant blocked in the conversion of indole to tryptophan, but not one blocked earlier in the tryptophan pathway, was resistant to 5FI. This is consistent with the hypothesis that 5FI was converted in vivo to 5FT which inhibited growth.     

Multiple Versions of the a Mating Type Locus of Coprinus Cinereus Are Generated by Three Paralogous Pairs of Multiallelic Homeobox Genes

The A mating type locus of Coprinus cinereus determines mating compatibility by regulating essential steps in sexual development. Each A locus contains several genes separated into two functionally independent complexes termed Aα and Aβ, and the multiple alleles of these genes generate an estimated 160 A mating specificities. The genes encode two classes of homeodomain-containing proteins designated HD1 and HD2. In this report we describe two newly cloned loci, A2 and A5, and compare them with A42, A43 and A6 that we have described previously. An Aβ-null locus, retaining just a single active HD1 gene from the α-complex, was generated by mutation. Using this as a transformation host, gene combinations that promote A-regulated development were identified. We demonstrate that each A locus contains members of three paralogous pairs of HD1 and HD2 genes. Different allelic versions of gene pairs are compatible but paralogous genes are incompatible. The genes present in four uncloned A loci were deduced using Southern analyses and transformations with available cloned genes. The combined analysis of nine A factors identifies sufficient A gene alleles to generate at least 72 A mating specificities.     

Mating Type and Sporulation in Yeast I. Mutations Which Alter Mating-Type Control over Sporulation

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant αα and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32°, but not at 23°. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.     

Light and Temperature Requirements during Fruit-Body Development of a Basidiomycete Mushroom, Coprinus congregates

Light initiated fruit body primordia of Coprinus congregatus Bull, ex Fr. grown at relatively high temperature (25°C) require a single dark period or low-temperature induction for completion of fruit-body development. The dark period requirement varied with the temperature regime during the inductive dark period A minimum requirement of 2.5 h was found al 15–20°C. Darkness always promotes development of fruit-body primordial, but cannot, be regarded as an absolute necessity until temperature exceeds about 17.5°C. Normal development of me primordia without darkness was obtained by lowering the temperature to 10°C for 6 h. It appeared that at high temperatures two successive stimuli were required for basidiocarp maturation, a light-off and a subsequent signal light-on signal. On the contrary, at 10°C a single low-temperature signal seemed to be involved. Thus, induction of fruit-body development could be produced by alternative pathways. These developmental features have been extended to other fungi and compared with the flowering processes of some short-day plants.     

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.     

Relationship between Monokaryotic Growth Rate and Mating Type in the Edible Basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBα and matBβ) and to correlate the presence of specific matBα and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

Frequent Changes in the Number of Reiterated Ribosomal RNA Genes Throughout the Life Cycle of the Basidiomycete Coprinus Cinereus

We have examined the stability of the tandemly repeated genes that encode the ribosomal RNA in Coprinus cinereus. These genes are contained within two linked HindIII fragments in a 3.0-Mb chromosome. We monitored the size of these fragments in both mitotic and meiotic segregants using the contour-clamped homogeneous electric field (CHEF) method. No length changes were observed in the smaller HindIII fragment (100 kb; 10 repeats) among the DNAs prepared from 46 asexual spore derivatives (oidia) or 128 meiotic segregants (basidiospores from 32 tetrads). However, the larger HindIII fragment (1100 kb; 120 repeats) did exhibit variability. Substantial changes, involving up to 40% of the larger HindIII fragment were recorded in 7 of 46 oidial isolates (including 4 of 22 transformed derivatives). To learn if the changes were confined to the vegetative portion of the life cycle, we examined transmission of HindIII variants through three crosses. In the first two crosses (16 tetrads total), no changes were observed in the large HindIII fragment. However, in the third cross (16 tetrads), each tetrad showed at least one alteration. In half of the tetrads from the third cross, the altered patterns segregated 2:2, suggesting that the changes occurred after mating but prior to premeiotic DNA replication. We conclude that breakage and rejoining reactions within the rDNA are frequent and are not confined to any particular stage of the life cycle. It also appears that certain repeats are sheltered from these events. Finally, marked differences in rDNA stability were observed in the crosses analyzed.     

DNA-mediated transformation of the basidiomycete Coprinus cinereus.

We have developed a simple and efficient transformation system for the agaric fungus, Coprinus cinereus. Protoplasts were prepared from asexual spores that harbor one or two mutations in the structural gene for tryptophan synthetase. The protoplasts can be stably transformed using the cloned Coprinus gene at a frequency of 1 in 10(4) viable protoplasts. A variety of molecular events accompanies the formation of stable transformants, including insertion of the transforming DNA at the homologous locus. The transforming DNA is stable through cell division, mating, fruiting body formation, and meiosis.     

Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus

Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi ( Agaricomycetes ) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5 , functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae . Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an α-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes δ-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of α-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated α-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.     

Inheritance of DNA methylation in Coprinus cinereus.

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.