Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes

Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H₂O₂ (2 mM) for optimum activity. The apparent K_m values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H₂O₂ were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.     

Expression of Proteins and Glycoproteins Encoded by the Haploid Nuclei in the Dikaryotic State in the Basidiomycete Agrocybe aegerita

The total proteins and concanavalin A-binding glycoproteins of the cultivated mushroom Agrocybe aegerita were studied in homokaryotic siblings and in dikaryotic strains. The glycoproteins exhibited considerable variability compared with the proteins; the genetic diversity detected in homokaryons in the glycoprotein analysis was 30-fold higher than the genetic diversity revealed by protein analysis, and the glycoprotein patterns could be used to characterize individual genotypes. We found that the expression of glycoproteins in haploid nuclei was significantly asymmetric when the nuclei were paired in dikaryons. The expression levels of the two component nuclei depended on their genotypes, and each haploid nucleus was characterized by its level of expression. Furthermore, some specific glycoproteins that were not detected in all of the homokaryons were newly synthesized in the dikaryotic strains. Among these was a glycoprotein designated gpAa-65, which was identified in all of the dikaryotic strains and appeared to be a good molecular marker of the dikaryotic state.     

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.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

Differences in Mitochondrial Genome Organization of Cryptococcus Neoformans Strains

The organization of the mitochondrial genomes in two strains belonging in different varieties of Cryptococcus neoformans was analysed. Physical maps of the mtDNA of the IFM5844 (var. neoformans) and IFO410 (var. grubii) strains were constructed by using EcoRI and EcoRV restriction enzymes; functional maps were constructed by hybridization, cloning and sequencing. Most of the genes important in the mitochondrial function (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, ATP6, ATP9, COX1, COX2 and COB) and protein synthesis (SsrRNA and LsrRNA) were localized. We did not find any differences between the strains in the order of these genes. However, they differed significantly in the sizes of the mtDNAs: 32.6 kb for IFM5844, and 24.1 kb for IFO410. This can be attributed to two large regions of the mtDNA. In these regions, differences were found in the numbers of introns in COX1 (no intron in var. grubii, 5 introns in var. neoformans), COB (1 intron in var. grubii, 2 introns in var. neoformans), LsrRNA (no intron in var. grubii, 2 introns in var. neoformans), and ND5 (no intron in var. grubii, 1 intron in var. neoformans) genes. In several introns of the COB and COX1 genes LAGLIDADG motifs were found. Differences were also observed in the nucleotide sequences of some genes and in the sizes and sequences of intergenic regions. The nucleotide sequences of the genes of the IFM and IFO strains were compared with those of the H-99 and JEC 21 strains from the database. Surprisingly high similarities were found between the strains belonging in var. grubii (IFO 410 and H-99) and var. neoformans (IFM 5844 and JEC 21).     

Suppression of Repeat-Mediated Gross Mitochondrial Genome Rearrangements by RecA in the Moss Physcomitrella patens

RecA and its ubiquitous homologs are crucial components in homologous recombination. Besides their eukaryotic nuclear counterparts, plants characteristically possess several bacterial-type RecA proteins localized to chloroplasts and/or mitochondria, but their roles are poorly understood. Here, we analyzed the role of the only mitochondrial RecA in the moss Physcomitrella patens. Disruption of the P. patens mitochondrial recA gene RECA1 caused serious defects in plant growth and development and abnormal mitochondrial morphology. Analyses of mitochondrial DNA in disruptants revealed that frequent DNA rearrangements occurred at multiple loci. Structural analysis suggests that the rearrangements, which in some cases were associated with partial deletions and amplifications of mitochondrial DNA, were due to aberrant recombination between short (<100 bp) direct and inverted repeats in which the sequences were not always identical. Such repeats are abundant in the mitochondrial genome, and interestingly many are located in group II introns. These results suggest that RECA1 does not promote but rather suppresses recombination among short repeats scattered throughout the mitochondrial genome, thereby maintaining mitochondrial genome stability. We propose that RecA-mediated homologous recombination plays a crucial role in suppression of short repeat-mediated genome rearrangements in plant mitochondria.     

火菇属野生菌株发酵液对栽培菌株的化感效应

为探究火菇属Flammulina野生菌株发酵液对栽培菌株的影响,以火菇属野生柳生金针菇F. rossica F52、野生金针菇F. filiformis F39和栽培金针菇F. filiformis F47为供试菌株,采用固体平板培养和液体发酵的方法,对比了添加不同体积分数火菇属野生菌株发酵液对栽培菌株平板中菌丝长速、液体发酵中菌丝生物量、干质量及漆酶和淀粉酶活力的影响。结果表明：不同体积分数野生金针菇菌株F39发酵液对栽培菌株F47菌丝长速有抑制作用,对发酵液中菌丝生物量无显著影响,当添加发酵液体积分数为50%时可显著提高菌株F47发酵液中漆酶活力,当体积分数为40%时,可显著提高菌株F47发酵液中淀粉酶的活力（P<0.05）;添加不同体积分数的野生柳生金针菇菌株F52发酵液对菌株F47除淀粉酶外其他指标均影响显著,添加体积分数为30%的发酵液对栽培金针菇F47菌丝长速具有显著的促进作用,当添加发酵液体积分数为40%时,对菌株F47液体发酵中菌丝干质量和漆酶活力均有显著促进作用（P<0.05）。证明了化感效应不仅存在于属间,在同属下的种间也存在。     

Protein mapping and genome expression variations in the basidiomycete Agrocybe aegerita

Summary A procedure suitable for the extraction and mapping of total proteins from the basidiomycete, Agrocybe aegerita, was developed. A. aegerita mycelia were fragmented either with a Dangoumeau grinder, an X-press bomb or a sonicator and the efficiency of these three disruption methods were compared. The extraction buffer composition was optimized to avoid proteolytic activities. 2D-SDS-PAGE analysis of protein extracts showed that the rate of reproducibility depending on extractions and electrophoretic separations was always greater than 96% for all strains. The differences in efficiency observed between the breaking procedures indicate that the A. aegerita cell wall is more mechanically resistant than that of other basidiomycetes. The efficient action of protease inhibitors (PMSF and SDS) showed that A. aegerita mycelia contains numerous and/or highly active proteases. Reproducibility of protein extraction and separation methods allowed the establishment and the comparison of standard maps. Qualitative and quantitative variations in gene products between a wild dikaryotic strain and 11 homokaryotic strains from its progeny were examined. The genetic diversity, determined by comparing the distribution of proteic variations in 11 homokaryons from the same progeny, was comparable to that observed between co-isogenic homokaryons of another basidiomycete.     

Simple Colorimetric Method for Detecting Degenerate Strains of the Cultivated Basidiomycete Flammulina velutipes (Enokitake)

Degeneration of cultivated strains of Flammulina velutipes is a serious problem. We developed a simple colorimetric method to detect degenerate strains by using a liquid medium supplemented with bromothymol blue and lactose. The ability of a strain to develop normal mushrooms could be determined by the color of the medium.     

Genetic evidence for nonrandom sorting of mitochondria in the basidiomycete Agrocybe aegerita.

We studied mitochondrial transmission in the homobasidiomycete Agrocybe aegerita during plasmogamy, vegetative growth, and basidiocarp differentiation. Plasmogamy between homokaryons from progeny of three wild-type strains resulted in bidirectional nuclear migration, and the dikaryotization speed was dependent on the nuclear genotype of the recipient homokaryon. Little mitochondrial migration accompanied the nuclear migration. A total of 75% of the dikaryons from the fusion lines had both parental mitochondrial haplotypes (mixed dikaryons), and 25% had only a single haplotype (homoplasmic dikaryons); with some matings, there was a strong bias in favor of one parental haplotype. We demonstrated the heteroplasmic nature of mixed dikaryons by (i) isolating and subculturing apical cells in micromanipulation experiments and (ii) identifying recombinant mitochondrial genomes. This heteroplasmy is consistent with the previously reported suggestion that there is recombination between mitochondrial alleles in A. aegerita. Conversion of heteroplasmons into homoplasmons occurred (i) during long-term storage, (ii) in mycelia regenerated from isolated apical cells, and (iii) during basidiocarp differentiation. Homokaryons that readily accepted foreign nuclei were the most efficient homokaryons in maintaining their mitochondrial haplotype during plasmogamy, long-term storage, and basidiocarp differentiation. This suggests that the mechanism responsible for the nonrandom retention or elimination of a given haplotype may be related to the nuclear genotype or the mitochondrial haplotype or both.     

Genetic variability of the wild incompatibility alleles of the tetrapolar basidiomycete Agrocybe aegerita

Summary The variability of the sexual incompatibility genes of Agrocybe aegerita was investigated in the homokaryotic progeny of 13 wild dikaryotic strains from five distinct European geographic origins. Results of mating tests allowed identification of 18 A alleles and 16 B alleles out of a possible 26 different alleles for each in the sample. The determination and the comparison by a contingency ² test of the frequencies of allele replications between intra- and interregional matings showed no departure from a random distribution of incompatibility alleles. The allelic series estimated for the incompatibility genes of the entire population of A. aegerita, 30 A and 25 B aleles, are significantly less extensive than those already hypothesized for other tetrapolar hymenomycetes. However, the low variability of incompatibility genes has little effect on the outbreeding efficiency (92.6%) of this mushroom. The low variability of the incompatibility alleles and the apparent absence of intrafactorial recombination could relate to a single-locus structure of the incompatibility genes in A. aegerita.     

The haloperoxidase of the agaric fungus Agrocybe aegerita hydroxylates toluene and naphthalene

The mushroom Agrocybe aegerita secretes a peroxidase (AaP) that catalyzes halogenations and hydroxylations. Phenol was brominated to 2- and 4-bromophenol (ratio 1:4) and chlorinated to a lesser extent to 2-chlorophenol. The purified enzyme was found to oxidize toluene via benzyl alcohol and benzaldehyde into benzoic acid. A second fraction of toluene was hydroxylated to give p-cresol as well as o-cresol and methyl-p-benzoquinone. The UV-Vis absorption spectrum of purified AaP showed high similarity to a resting state cytochrome P450 with the Soret band at 420 nm and additional maxima at 278, 358, 541 and 571 nm; the AaP CO-complex had a distinct absorption maximum at 445 nm that is characteristic for heme-thiolate proteins. AaP regioselectively hydroxylated naphthalene to 1-naphthol and traces of 2-naphthol (ratio 36:1). H2O2 was necessarily required for AaP function and hence the hydroxylations catalyzed by AaP can be designated as peroxygenation and the enzyme as an extracellular peroxygenase.     

Molecular character of the recombinant antitumor lectin from the edible mushroom Agrocybe aegerita

The lectin from Agrocybe aegerita (AAL) has been found to possess potent tumor-suppressing function and tumor cell apoptosis-inducing activity. In this paper, we report the full sequence, the active expression of the gene encoding AAL at a high level and bioassay of the binding property with lactose, apoptosis-inducing activity and DNase activity of recombinant AAL (rAAL). The results reveal that AAL is a member of the galectin family and the dimeric form is the active unit for the functional performance. The rAAL showed comparable tumor cell apoptosis-inducing activity with the wild AAL but no DNase activity at all. The molecular characters revealed by this study are significant for the in-depth investigation of the functional mechanism of this interesting protein.     

High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces

Current genome editing techniques available for Saccharomyces yeast species rely on auxotrophic markers, limiting their use in wild and industrial strains and species. Taking advantage of the ancient loss of thymidine kinase in the fungal kingdom, we have developed the herpes simplex virus thymidine kinase gene as a selectable and counterselectable marker that forms the core of novel genome engineering tools called the Haploid Engineering and Replacement Protocol (HERP) cassettes. Here we show that these cassettes allow a researcher to rapidly generate heterogeneous populations of cells with thousands of independent chromosomal allele replacements using mixed PCR products. We further show that the high efficiency of this approach enables the simultaneous replacement of both alleles in diploid cells. Using these new techniques, many of the most powerful yeast genetic manipulation strategies are now available in wild, industrial, and other prototrophic strains from across the diverse Saccharomyces genus.     

Position and Content Paradigms in Genome Rearrangements: The Wild and Crazy World of Permutations in Genomics

Modellers of large-scale genome rearrangement events, in which segments of DNA are inverted, moved, swapped, or even inserted or deleted, have found a natural syntax in the language of permutations. Despite this, there has been a wide range of modelling choices, assumptions and interpretations that make navigating the literature a significant challenge. Indeed, even authors of papers that use permutations to model genome rearrangement can struggle to interpret each others’ work, because of subtle differences in basic assumptions that are often deeply ingrained (and consequently sometimes not even mentioned). In this paper, we describe the different ways in which permutations have been used to model genomes and genome rearrangement events, presenting some features and limitations of each approach, and show how the various models are related. This paper will help researchers navigate the landscape of permutation-based genome rearrangement models and make it easier for authors to present clear and consistent models.     

The Entire Mitochondrial Genome of Macrophthalmus abbreviatus Reveals Insights into the Phylogeny and Gene Rearrangements of Brachyura

Brachyuran crabs comprise the most species-rich clades among extant Decapoda and are divided into several major superfamilies. However, the phylogeny of Brachyuran remains controversial, comprehensive analysis of the overall phylogeny is still lacking. Complete mitochondrial genome (mitogenome) can indicate phylogenetic relationships, as well as useful information for gene rearrangement mechanisms and molecular evolution. In this study, we firstly sequenced and annotated the complete mitogenome of Macrophthalmus abbreviatus (Brachyura; Macrophthalmidae). The mitogenome length of M. abbreviatus is 16,322 bp, containing the entire set of 37 genes and a control region typically observed in Brachyuran mitogenomes. The genome composition of M. abbreviatus was highly A+T biased 76.3% showing positive AT-skew (0.033) and negative GC-skew (??0.351). In M. abbreviatus mitogenome, most tRNA genes were folded into the clover-leaf secondary structure except trnH, trnS1 and trnC, which was similar to the other species in Macrophthalmidae. Phylogenetic analysis showed that all families form a monophyletic, and Varunidae and Macrophthalmidae clustered into a monophyletic clade as sister groups. Comparative analyses of rearrangement among Brachyura revealed that Varunidae (Grapsoidea) and Macrophthalmidae (Ocypodoidea) had the same gene order, which reinforced the result of phylogeny. The combined results of two aspects revealed that the polyphyly of Ocypodoidea and Grapsoidea were well supported. In general, the results obtained in this research will contribute to further studies on molecular based for the classification and gene rearrangements of Macrophthalmidae or even Brachyura.

     

Distribution of Brown Blotch Bacteria in Wild and Cultivated Species of Basidiomycetes

Wild and cultivated Basidiomycetes species were cultured to determine the distribution of bacteria causing brown blotch disease of Agaricus bisporus. Colonies from each basidiocarp were screened for brown blotch organisms by the white line and host pathogenicity tests. Isolates causing brown blotch were identified as Pseudomonas tolaasi and an Arthrobacter species.     

Transgenerational Inheritance of Diet-Induced Genome Rearrangements in Drosophila

Ribosomal RNA gene (rDNA) copy number variation modulates heterochromatin formation and influences the expression of a large fraction of the Drosophila ge-nome. This discovery, along with the link between rDNA, aging, and disease, high-lights the importance of understanding how natural rDNA copy number variation arises. Pursuing the relationship between rDNA expression and stability, we have discovered that increased dietary yeast concentration, emulating periods of dietary excess during life, results in somatic rDNA instability and copy number reduction. Modulation of Insulin/TOR signaling produces similar results, indicating a role for known nutrient sensing signaling pathways in this process. Furthermore, adults fed elevated dietary yeast concentrations produce offspring with fewer rDNA copies demonstrating that these effects also occur in the germline, and are transgenera-tionally heritable. This finding explains one source of natural rDNA copy number variation revealing a clear long-term consequence of diet.