Kiminori Shimizu

Publications (17)13.44 Total impact

• Article: Non-homologous end joining pathway of the human pathogen Cryptococcus neoformans influences homologous integration efficiency but not virulence

  Hao-Man Li, Eric V. Virtudazo, Akio Toh-e, Masashi Yamaguchi, Susumu Kawamoto, Kiminori Shimizu
  [show abstract] [hide abstract]
  ABSTRACT: The efficiency of gene targeting by integration through homologous recombination (homologous integration, HI) in the human pathogen Cryptococcus neoformans remains unsatisfactory. In order to achieve a much more efficient gene targeting system in C. neoformans, a new double knockout strain in genes involved in the non-homologous end joining (NHEJ) pathway was constructed. HI frequency was elevated by as much as approximately fivefold in the single or double knockout strains in NHEJ genes, and the frequency depended on the gene targeted. None of the NHEJ gene knockouts showed significant differences in regular growth, sensitivity to DNA-damaging drugs or UV, and virulence compared to the wild-type control, suggesting that the NHEJ pathway does not play a significant role in these biological stresses in C. neoformans. It was also suggested that the genes analyzed in this study are components of a single NHEJ pathway, as the mutants (including the double mutant) displayed the same phenotypes. KeywordsDNA ligase IV-Gene manipulation-KU protein-Molecular genetics
  Mycoscience 04/2012; 51(4):272-280. · 1.21 Impact Factor
• Article: Deletion of CnLIG4 DNA ligase gene in the fungal pathogen Cryptococcus neoformans elevates homologous recombination efficiency

  Kiminori Shimizu, Hao-Man Li, Eric V. Virtudazo, Akira Watanabe, Katsuhiko Kamei, Masashi Yamaguchi, Susumu Kawamoto
  [show abstract] [hide abstract]
  ABSTRACT: In eukaryotes from yeasts to human, DNA double-strand breaks are repaired by nonhomologous end-joining (NHEJ) or homologous integration (HI). In the human pathogenic yeast Cryptococcus neoformans, gene manipulation by HI does not occur frequently because ectopic integration by NHEJ is predominant, and it has been necessary to screen 30–100 transformants per experiment to obtain transformants with the desired genotypes. To overcome this problem, we constructed a strain in which one of the NHEJ-related genes, CnLIG4, was deleted. CnLIG4 encodes a homologue of the human DNA ligase IV involved in the last step of DNA repair by NHEJ. Gene targeting in the URA5 locus of a URA5-lacking strain TAD1 with URA5 gene fragments having 1-kb flanking sequences achieved 80% HI efficiency, which is higher than that of the wild-type control (50%). Growth phenotypes and virulence were not attenuated by deletion of the CnLIG4 gene. Such results suggest that the CnLIG4 knockout strain created in this study provides an additional alternative for the molecular genetics study of C. neoformans.
  Mycoscience 04/2012; 51(1):28-33. · 1.21 Impact Factor
• Article: ChMCO1 of Cochliobolus heterostrophus is a new class of metallo-oxidase, playing an important role in DHN-melanization

  Yoshimoto Saitoh, Kosuke Izumitsu, Atsushi Morita, Kiminori Shimizu, Chihiro Tanaka
  [show abstract] [hide abstract]
  ABSTRACT: A metallo-oxidase gene from a phytopathogenic filamentous fungus, Cochliobolus heterostrophus was cloned. Structural prediction of ChMco1 indicated that this protein lacks a transmembrane helix and is soluble, whereas other known fungal metallo-oxidases including Saccharomyces cerevisiae FET3 are localized to the cell membrane. The results of searches in fungal genomic databases and phylogenetic analysis of fungal metallo-oxidases revealed that ChMco1 and its allies are distinct homologues of Fet3 and unique to filamentous ascomycetous species including C. heterostrophus. We performed a functional analysis of ChMCO1 by generating null mutants for the ChMco1 gene. The ChMco1 null (∆ChMco1) mutants clearly had reduced melanization, although they showed normal growth and conidiation. Results also show that ∆ChMco1 mutants lost laccase activity. These results suggest that ChMCO1 is a novel class of metallo-oxidase that is necessary for laccase activity and melanization. KeywordsFilamentous fungi-Laccase-Mco1-Melanin
  Mycoscience 04/2012; 51(5):327-336. · 1.21 Impact Factor
• Article: Coprinopsis neophlyctidospora sp. nov., a new ammonia fungus from boreal forests in Canada

  JAY K. RAUT, AKIRA SUZUKI, TOSHIMITSU FUKIHARU, KIMINORI SHIMIZU, SUSUMU KAWAMOTO, CHIHIRO TANAKA
  [show abstract] [hide abstract]
  ABSTRACT: Coprinopsis neophlyctidospora sp. nov. (Basidiomycota, Agaricales), collected in urea treated soil of boreal forests from Canada is described and illustrated. Its micromorphological features, phylogenetic analysis, and mating test delineate this taxon as a new species. In addition, its ecological characters also indicate it is a new ammonia fungus.
  Mycotaxon. 01/2011; 115:227-238. doi: 10.5248/115.227.
• Article: [Phosphate regulation in Cryptococcus neoformans].

  Akio Toh-e, Kiminori Shimizu, Hao-Man Li, Masafumi Nishizawa, Susumu Kawamoto
  Nippon Ishinkin Gakkai Zasshi 01/2011; 52(1):19-23.
• Article: Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata.

  Yuichi Namiki, Keigo Ueno, Hiroki Mitani, Eric V Virtudazo, Misako Ohkusu, Kiminori Shimizu, Susumu Kawamoto, Hiroji Chibana, Masashi Yamaguchi
  [show abstract] [hide abstract]
  ABSTRACT: Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10 min, filamentous materials covered the entire surface of the protoplast at 1 h, granular materials started filling interspaces of filamentous materials at 2 h and regeneration was completed at 6 h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40 nm, and composed of β-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.
  Journal of electron microscopy 01/2011; 60(2):157-65. · 1.31 Impact Factor
• Conference Proceeding: A new record of an ammonia fungus, Coprinopsis rugosobispora collected from urea-treated boreal forest in Canada

  O Jay Kant Raut, Akira Suzuki, Toshimitsu Fukiharu, Kiminori Shimizu, Chihiro Tanaka, Shogo Takeshige
  [show abstract] [hide abstract]
  ABSTRACT: Addition of ammonia or any nitrogenous materials to the soil that release ammonia casusing alkaline condition during decomposition stimulates the fruiting of a particular chemoechological group of fungi, called ammonia fungi (Sagara 1975). The study of ammonia fungi by artificial application of urea in forest soil has been done in diverse geographical regions such as in Japan, Taiwan, New Zealand, Western Australia, UK and western US. Up to date about 70 species of ammonia fungi have been recorded in those regions. However, ammonia fungi in boreal forest of American continent have not yet been investigated. Thus, we collected the soils of A0 and the upper layer of HA horizons in plant pots from aspen forest near Edmonton, Canada. Thereafter, we applied urea (granular fertilizer; 46% nitrogen, 10 mg/g dry soil) in plant pots and incubated at 25˚C under 12 hours dark and light regime. After 40 days of incubation, several baisdiomata of Coprinopsis species appeared. Among them one specimen was identified as C. rugosobispora based on macro- and microscopic features. Morphologically this species is very similar to C. phlyctidospora which is characterized by warty, ovoid basidiospores, and diverticulate veil elements. C. phlyctidospora has 4-spored basidia while C. rusgosobispora has only 2-spored. In the beginning, it was thought probably it is only a 2-spored form of that species, but based on the basidiospore size (9.8-11.7 ×8.3-9.6 µm) which is distinctly larger than in the 4-spored (8.4-10.6×6.0-7.6 µm). It was therefore separated from the C. phlyctidospora. Furthermore in this study we investigated its phylogenetic relationship based on the nuclear rDNA sequence in ITS regions and mating reactions among its close allies and further confirmed it as a distinct species This is the first record of C. rusgosobispora from American continent since it has been collected only from Europe(Belgium and the Netherland). Although urea effectively stimulated its occurrence but it has not yet been reported any other urea application studies so far. This indicates it is a new record in ammonia fungi as well.
  The 6th meeting of East Asia for collaboration on Edible Fungi, Gwangju, Korea; 01/2010
• Source

  Available from: Eiji Tanaka

  Article: Isolation of basidiomycetous anamorphic yeast-like fungus Meira argovae found on Japanese bamboo

  Eiji Tanaka, Kiminori Shimizu, Yumi Imanishi, Fumitoshi Yasuda, Chihiro Tanaka
  [show abstract] [hide abstract]
  ABSTRACT: A basidiomycetous anamorphic yeast-like fungus, isolated from new bamboo shoots collected in Japan, was assigned to Meira argovae by comparison of conidial morphology, physiological characteristics, rDNA sequences, and DNA-DNA relatedness with the ex-type strains of Meira species. This is the first record of the finding of M. argovae from other than mite cadavers and in regions other than Israel. Phylogenetic analysis based on the D1-D2 domain demonstrated that Meira species and teleomorphic Dicellomyces species, which include a bamboo leaf parasite, D. gloeosporus, formed sister clades.
  Mycoscience 09/2008; 49(5):329-333. · 1.21 Impact Factor
• Source

  Available from: jsmm.org

  Article: Construction of a complete URA5 deletion strain of a human pathogenic yeast Cryptococcus neoformans.

  Antra Drivinya, Kiminori Shimizu, Kanji Takeo
  [show abstract] [hide abstract]
  ABSTRACT: Cryptococcus neoformans is an opportunistic human pathogen, which infects the central nervous system causing the fatal disease, meningitis. In order to understand the genetic background of this human pathogen, the basic molecular manipulation techniques of deletion, overexpression, and so on have been developed. URA5, a gene encoding orotate phosphoribosyltransferase, has frequently been used to introduce foreign gene fragments by complementing ura5 mutant strains, which are not, however, stable; reversion to uracil prototroph is thus frequently observed on selective condition. The high possibility of reversion makes it inconvenient to use this mutation to identify appropriate transformants and thus, manipulation in molecular genetics. We report here the isolation of a stable ura5 mutant of C. neoformans, designated as TAD1, by eliminating the URA5 gene by homologous recombination using the biolistic DNA delivery system. The availability of the stable ura5 mutant offers the advantage that no spontaneous reversion occurs so that a satisfactory rate of homologous recombination can be achieved. The strain will allow efficient genomic analysis in C. neoformans.
  Nippon Ishinkin Gakkai Zasshi 02/2004; 45(1):1-6.
• Source

  Available from: Eiji Tanaka

  Article: Species separation in Curvularia “geniculata” group inferred from Brn1 gene sequences

  Guangyu Sun, Shinichi Oide, Eiji Tanaka, Kiminori Shimizu, Chihiro Tanaka, Mitsuya Tsuda
  [show abstract] [hide abstract]
  ABSTRACT:  Brn1, a reductase gene involved in the melanin biosynthetic pathway, was adopted for species delimitation among members in the “geniculata” group of Curvularia species and proved to be useful for this purpose. Phylogenetic trees of these fungal members were constructed from nucleotide sequences of this region. The so-called geniculata group of Curvularia was separated into several clusters. The conidial morphology of the members in each cluster is closely similar but clearly different among discrete clusters. The phylogenetic groups almost concurred with the morphological grouping. Thus, the synonymous treatment of Curvularia affinis, C. fallax, and C. senegalensis to C. geniculata in a previous study was supported. The isolates with warping hilum conidia were clearly different from C. geniculata and separated into two clusters. C. geniculata ATCC 6671 made an independent cluster situated near these clusters. The protuberant hilum species were located separately in the phylogenetic trees. For sound taxonomic treatment of these isolates, we should accumulate more information and retain our species determination for them.
  Mycoscience 05/2003; 44(3):239-244. · 1.21 Impact Factor
• Article: Polyoxin Resistance of Reddish Brown Laboratory Mutants of Cochliobolus heterostrophus

  Chihiro TANAKA, Kiminori SHIMIZU, Abdul GAFUR, Mitsuya TSUDA
  [show abstract] [hide abstract]
  ABSTRACT: Six reddish brown polyxin-resistant mutants of Cochliobolus heterostrophus were isolated after ethyl methanesul-phonate and N-nitroquinoline oxide mutageneses followed by selection on polyoxin. All the mutants were highly resistant to polyoxin (MIC > 1600 μg/ml). When mutants were crossed with the wild-type strain, all crosses had a 1 : 1 ratio of mutant (reddish brown pigmentation and polyoxin resistance) : wild type (non-reddish brown pigmentation and polyoxin sensitivity), indicating that the phenotypes in these strains were due to alteration at a single gene locus in each strain. Allelism tests revealed the existence of two loci, Pol2 and Pol5. The results of the crossing and mutation-rate studies suggest that the each gene was pleiotropic for the reddish brown color and polyoxin resistance.
  Journal of General Plant Pathology 04/2002; 68(2):141-146. · 0.69 Impact Factor
• Article: Phylogeny of Bipolaris inferred from nucleotide sequences of Brn1, a reductase gene involved in melanin biosynthesis.

  Kiminori Shimizu, Chihiro Tanaka, You-Liang Peng, Mitsuya Tsuda
  [show abstract] [hide abstract]
  ABSTRACT: The Brn1 reductase melanin biosynthesis gene in the fungal genus Bipolaris was sequenced in 74 strains of 22 species. The Brn1 region was highly conserved among the species examined at the nucleotide and the amino acid levels. To elucidate the phylogenetic relationships among Bipolaris species, trees were inferred from nucleotide sequences of this region. Species in these trees formed exclusive clusters clearly separated from one another, except for B. panici-miliacei and B. setariae, and B. victoriae and B. zeicola. When unidentified strains were added to this tree, they fell within known species or formed independent clusters. These data indicated that the Brn1 gene region was suitable for species-level systematics within the genus. The results also suggest that Bipolaris consists of two or more clades that may reflect teleomorphic connections.
  The Journal of General and Applied Microbiology 09/1998; 44(4):251-258. · 0.98 Impact Factor
• Article: Molecular analysis and characterization of the Cochliobolus heterostrophus beta-tubulin gene and its possible role in conferring resistance to benomyl.

  Abdul Gafur, Chihiro Tanaka, Kiminori Shimizu, Seiji Ouchi, Mitsuya Tsuda
  [show abstract] [hide abstract]
  ABSTRACT: Cochliobolus heterostrophus Tub1 described here is the first beta-tubulin gene characterized from a naturally occurring benomyl-resistant ascomycete plant pathogen. The gene encodes a protein of 447 amino acids. The coding region of Tub1 is interrupted by three introns, of 116, 55, and 56 nt, situated after codons 4, 12, and 53, respectively. As a result of the preference for pyrimidines in the third position of the codons when a choice exists between purines and pyrimidines, codon usage in the Tub1 gene is biased. Tub1 shows high homology with beta-tubulin genes of other ascomycete species. However, Tub1 is exceptional in having Tyr(167), compared with Phe(167), possessed by beta-tubulin genes of other ascomycetes sequenced thus far. The Tyr(167) residue has been associated with benomyl resistance in other organisms. In contrast, all other benomyl-implicated residues of Tub1 correspond to sensitivity. Based on these results, we suggest that benomyl resistance in the fungus probably is attributed to Tyr(167).
  The Journal of General and Applied Microbiology 07/1998; 44(3):217-223. · 0.98 Impact Factor
• Article: Genetic analysis of Cochliobolus heterostrophus polyoxin-resistant mutants

  Abdul Gafur, Chihiro Tanaka, Kiminori Shimizu, Seiji Ouchi, Mitsuya Tsuda
  [show abstract] [hide abstract]
  ABSTRACT: Nine polyoxin-resistant mutants ofCochliobolus heterostrophus were isolated after ethyl methanesulphonate mutagenesis. All were highly resistant to polyoxin (MIC≥1,600 ppm). Crosses between the mutants and a wild-type strain revealed that the resistance trait was inherited to the offsprings in different fashions. Four of the mutant strains inherited polyoxin resistance in a 1∶1 segregation ratio, indicating that the phenotypes in these strains were due to alteration at a single locus. Allelism tests revealed four new loci,Pol1, Pol2, Pol3 andPol4, for polyoxin resistance in these mutant strains. The genes responsible for the phenotypes of the other five mutant strains were not determined, because of extremely slow growth of progenies in one cross, sterility in another cross, and inexplicable responses to polyoxin of the progenies in the other crosses. No linkage was detected between the genes for polyoxin resistance and mating type.
  Mycoscience 06/1998; 39(2):155-159. · 1.21 Impact Factor
• Article: Cloning of Brn1, a reductase gene involved in melanin biosynthesis in Cochliobolus heterostrophus.

  Kiminori Shimizu, Chihiro Tanaka, Mitsuya Tsuda
  [show abstract] [hide abstract]
  ABSTRACT: The plant pathogenic fungus Cochliobolus heterostrophus produces melanin, a black pigment, via 1, 8-dihydroxynaphthalene. The deficiency of C. heterostrophus Brn1(-) mutant was complemented with the cosmid clone pCOS/ML6, screened by heterologous hybridization with the genes involved in the melanin biosynthesis of Alternaria alternata. We determined the DNA sequence of the Brn1 gene and its flanking regions. The Brn1 gene contains one open reading frame consisting of three exons separated by two introns. A comparison of the nucleotide and predicted amino acid sequence of the Brn1 gene with those of other fungal reductase genes involved in melanin biosynthesis indicates significant similarity as well as the pathway of melanin biosynthesis.
  The Journal of General and Applied Microbiology 07/1997; 43(3):145-150. · 0.98 Impact Factor
• Article: Host Range of Aciculosporium take, the Causal Agent of Witches' Broom of Bamboo Plants

  Mitsuya Tsuda, Kiminori Shimizu, Keishi Matsumura, Eiji Tanaka, Chihiro Tanaka and Yoshimichi Doi
  Bulletin of the National Science Museum, Series B. 01/1997; 23:25-34.
• Article: Genetic analyses of Cochliobolus heterostrophus albino mutant with deficiencies at two loci

  Kiminori Shimizu, Chihiro Tanaka, Mitsuya Tsuda
  [show abstract] [hide abstract]
  ABSTRACT: A new class of albino mutant ofCochliobolus heterostrophus was isolated. Its colony color was indistinguishable from that of albino mutants previously reported. Application of the melanin intermediate scytalone induced this mutant to pigment slightly, but not completely. Genetic analyses showed that the mutant had two deficient genes. When only one of these genes was deficient, the colony color was indistinguishable from the wild type, whereas deficiency of both genes resulted in the albino phenotype. These deficiencies lie upstream of scytalone biosynthesis. These genes were designated asCal1 andCal2.
  Mycoscience 09/1996; 37(3):345-349. · 1.21 Impact Factor