Yutaka Kitamoto

Purification and properties of betaine aldehyde dehydrogenase from Xanthomonas translucens

Abstract Betaine aldehyde dehydrogenase from Xanthomonas translucens was purified to apparent homogeneity by ammonium sulfate fractionation, followed by ion-exchange, butyl-Toyopearl and gel filtration chromatography. The amino acid composition and the N-terminal sequence of 35 amino acid residues were determined. The enzyme was found to be a tetramer with identical 50 kDa subunits. Both NAD and NADP could be used as a cofactor for the enzyme and K m values for NAD and NADP were 70 μM and 50 μM, respectively. The enzyme was highly specific for betaine aldehyde and the K m value for betaine aldehyde was 0.19 mM.

Identification and linkage mapping of the genes for the putative homeodomain protein ( hox1 ) and the putative pheromone receptor protein homologue ( rcb1 ) in a bipolar basidiomycete, Pholiota nameko

In the current studies, we sequenced and characterized the gene for the homeodomain protein (hox1) in a bipolar mushroom, Pholiota nameko, which is a putative homologue of A mating type genes in the tetrapolar basidiomycete, Coprinopsis cinerea. We also sequenced and characterized the gene for the pheromone receptor (rcb1) in P. nameko, which is a putative homologue of the B mating type genes in C. cinerea. Restriction fragment length polymorphism (RFLP) and linkage analyses indicated that the both genes are present as a single locus on the different chromosome. Moreover, in P. nameko, the hox1 gene was mapped to the A mating type locus in linkage group I. However, rcb1 was not linked to the A mating type locus and was mapped to the other linkage group. These results strongly suggest that hox1 regulates with incompatibility in the bipolar mushroom, and that rcb1 may not affect the mating function in P. nameko. This is the first report regarding the structure of the mating type genes in bipolar mushrooms.

A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum

SummaryAn enzyme from Trichoderma harzianum dissolved the cell walls of a wide range of filamentous fungi belonging to Basidiomycotina, Ascomycotina, Deuteromycotina, and Zygomycotina and so could be used to make protoplasts. A lyophilized preparation of the Trichoderma enzyme had about 0.3 units/mg β-1,3-glucanase activity and 0.36 units/mg chitinase activity. About twice as many protoplasts were produced from different species of fungi by a single treatment with this enzyme than with combined commercial enzymes. The greatest number of protoplasts could be produced from most of the fungi by incubation for about 2 h t 30°C, but the number was decreased by incubation for more than 4 h or by use of a higher dose of the enzyme. An enzyme prepared by bentonite treatment from the original Trichoderma enzyme had less proteinase activity and protoplasts were fairly stable with this product during incubation for 8 h. Protoplasts produced by the proteinase-reduced preparation of the Trichoderma enzyme from three fungi regenerated at about 1.8 times the rate of those produced by the original enzyme.

A new method for the preservation of fungus stock cultures by deep-freezing

Abstract Recovery of 66 fungus stock cultures including Oomycota, Zygomycota, Ascomycota, Basidiomycota, and mitosporic mycetes were examined after cryopreservation. Almost all the stock cultures remained viable when the mycelia that had grown over the sawdust medium containing 10% glycerol as the cryoprotectant (65% moisture content, W/W) were frozen rapidly at −85°C and then allow to thaw naturally at room temperature. Test stock cultures were preserved for more than 10 years by this preservation method without any programmed precooling and rapid thawing for their cryopreservation. Most of the test fungi could survive for 5 years in medium containing 10% glycerol even after alternate freezing and thawing at intervals of 6 months. When a strain of Flammulina velutipes was tested for mycelial growth rate and productivity of fruit-bodies after cryopreservation for 3 years, the fungus reproduced with its initial capability. These results demonstrate that the sawdust-freezing method using a cryoprotectant is expected to be a reliable and easy preservation method for fungus stock cultures.

An action spectrum for photoinduction of pileus formation in a basidiomycete, Favolus arcularius.

SummaryThe action spectrum for photoinduction of pileus formation in Favolus arcularius (Fr.) Ames. shows four maxima, at ca. 375–385, 420, 446, and 480 nm, and minima near 365, 400, 435, and 470 nm. Wavelengths above 510 nm were ineffective. The maxima coincided with four of the six maxima for primordium formation of this fungus, but the two maxima for the latter around 398 and 514 nm could not be detected. The photosensitive region for the initiation of pileus formation was localized in the apical region of the stipes.

The biological species of oyster mushrooms (Pleurotus spp.) from Asia based on mating compatibility tests

Mating compatibility of 25 Pleurotus species collected mainly from Asia was tested by either the Mon–Mon mating or the Di–Mon mating tests. The results showed 5 intersterility groups (P. ostreatus, P. pulmonarius, P. cornucopiae, P. cystidiosus, and P. salmoneostramineus complexes) and 7 independent interincompatible species (P. calyptratus, P. corticatus, P. dryinus, P. eryngii, P. nebrodensis, P. smithii, and P. ulmarius). The P. ostreatus complex includes P. ostreatus, P. ostreatus var. columbinus, P. djamor, and P. flabellatus. The P. pulmonarius complex has 7 taxa: P. pulmonarius, P. eugrammus, P. eugrammus var. brevisporus, P. sajor-caju, P. sapidus, P. sp. florida, and P. opuntiae. The P. cornucopiae complex includes its variant P. cornucopiae var. citrinopileatus. The P. cystidiosus complex includes P. abalonus. The P. salmoneostramineus complex includes 3 pink-colored mushrooms: P. salmoneostramineus, P. ostreatoroseus, and P. rhodophyllus. According to mating compatibility tests, 12 biological species were identified from among 25 Pleurotus species.

Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR

ABSTRACT Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

Complete mitochondrial DNA sequence of the Japanese flying fish Cypselurus hiraii

In this study, to develop a technique that enables authentication of processed seafood, the complete nucleotide sequence of the mitochondrial genome for the Japanese flying fish Cypselurus hiraii was determined. Three segments spanning the entire genome were amplified using polymerase chain reaction, and products were subsequently used as templates for direct sequencing with 60 primers. The genome (16 528 base pairs) was found to contain the same 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as those found in other vertebrate mitochondrial genomes, with the gene order being identical to that typical of vertebrates. A major noncoding region between the tRNAPro and tRNAPhe genes (868 base pairs) appears to be the control (D-loop) region, as it has several conserved blocks characteristic of control regions.

Phylogenetic analysis of oyster mushrooms (Pleurotus spp.) based on restriction fragment length polymorphisms of the 5′ portion of 26S rDNA

Polymorphism analysis of the 5′ portion of 26S rDNA from 34 Pleurotus strains (12 intersterility groups) collected mainly from Asia was performed. By combining the restriction fragment length polymorphism (RFLP) patterns obtained from digestions with seven restriction enzymes, the 34 Pleurotus strains were assigned to 11 RFLP types. Ten RFLP patterns corresponded with biological species, but one pattern was found in intersterility groups I (P. ostreatus complex), II (P. pulmonarius complex), and VIII (P. eryngii). The phylogenetic tree suggests that Pleurotus species have evolved in two patterns based on 26S rDNA RFLP data. One major cluster comprising the “P. ostreatus clade” is separated by relatively short branches, suggesting that the P. ostreatus complex, the P. pulmonarius complex, P. eryngii, and P. nebrodensis (intersterility groups I, II, VIII, and IX, respectively), share a recent common ancestor. RFLP data did not distinguish the species in the intersterility groups I, II, and VIII. The other major cluster apparently divided into five sublevel clusters in early stages of evolution and these clades split into terminal nodes in late stages of evolution: the P. calyptratus-salmoneostramineus clade, the P. cornucopiae-ulmarius clade, the P. dryinus clade, the P. corticatus clade, and the P. cystidiosus-smithii clade.

Transcriptional regulation of two cellobiohydrolase encoding genes (cel1 and cel2) from the wood-degrading basidiomycete Polyporus arcularius

In the current studies, we sequenced and characterized the genomic and complementary deoxyribonucleic acid clones encoding the cellobiohydrolase encoding genes cel1 and cel2 of Polyporus arcularius. The predicted amino acid sequences of Cel1 and Cel2 are similar to glycosyl hydrolase family 7 and 6 proteins, respectively. The expression of cel1 and cel2 was induced by microcrystalline cellulose (Avicel) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a very low level of cel1 and cel2 transcription regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of cel1 and cel2 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.