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author:("Oka, takumi")
1.  gfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and A. fumigatus 
Molecular microbiology  2013;90(5):1054-1073.
The cell walls of filamentous fungi in the genus Aspergillus have galactofuranose-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan, and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple galactofuranose monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in galactofuranose (Galf) antigen biosynthesis. Disruption of gfsA reduced binding of β-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitro galactofuranose antigen synthase assay revealed that GfsA has β1,5- or β1,6- galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-D-galactofuranose as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature, and limited formation of conidia. Several gfsA orthologs were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β-galactofuranosyltransferase, which was shown to be involved in galactofuranose antigen biosynthesis of O-glycans in the Golgi.
doi:10.1111/mmi.12416
PMCID: PMC3907285  PMID: 24118544
galactofuranose; galactofuranosyltransferase; O-glycans; cell wall; Aspergillus
2.  Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA) 
The filamentous fungus Paecilomyces variotii NBRC 109023 (teleomorph: Byssochlamys spectabilis NBRC 109023) degrades formaldehyde at concentrations as high as 2.4 % (w/v). In many prokaryotes and in all known eukaryotes, formaldehyde degradation is catalyzed by S-hydroxymethylglutathione (S-HMGSH) dehydrogenase. We report here the isolation and characterization of the gene encoding S-HMGSH dehydrogenase activity in P. variotii. The 1.6-kb fldA gene contained 5 introns and 6 exons, and the corresponding cDNA was 1143 bp, encoding a 40-kDa protein composed of 380 amino acids. FldA was predicted to have 74.3, 73.7, 68.5, and 67.4 % amino acid identity to the S-HMGSH dehydrogenases of Hansenula polymorpha, Candida boidinii, Saccharomyces cerevisiae, and Kluyveromyces lactis, respectively. The predicted protein also showed high amino acid similarity (84∼86 %) to the products of putative fldA genes from other filamentous fungi, including Aspergillus sp. and Penicillium sp. Notably, the P. variotii fldA gene was able to functionally complement a Saccharomyces cerevisiae strain (BY4741 ∆sfa1) lacking the gene for S-HMGSH dehydrogenase. The heterologous expression construct rendered BY4741 ∆sfa1 tolerant to exogenous formaldehyde. Although BY4741 (parental wild-type strain) was unable to degrade even low concentrations of formaldehyde, BY4741 ∆sfa1 harboring Paecilomyces fldA was able to degrade 4 mM formaldehyde within 30 h. The findings from this study confirm the essential role of S-HMGSH dehydrogenase in detoxifying formaldehyde.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6203-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6203-8
PMCID: PMC4322224  PMID: 25398285
Paecilomyces variotii; Formaldehyde degradation; S-hydroxymethylglutathione dehydrogenase; fldA; Heterologous expression
3.  Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain 
Toxins  2014;6(6):1882-1895.
Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC50) of PS5 to MOLT-4 cells was approximately 0.075 μg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type β-pore-forming toxins (β-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others.
doi:10.3390/toxins6061882
PMCID: PMC4073135  PMID: 24945755
Bacillus thuringiensis; parasporin; cry toxin; β-pore-forming toxin
4.  Draft Genome Sequence of the Formaldehyde-Resistant Fungus Byssochlamys spectabilis No. 5 (Anamorph Paecilomyces variotii No. 5) (NBRC109023) 
Genome Announcements  2014;2(1):e01162-13.
Byssochlamys spectabilis no. 5 (anamorph Paecilomyces variotii no. 5) (NBRC109023) was isolated from a soil sample in 2001 in Kumamoto Prefecture, Japan. This fungus is highly resistant to formaldehyde. Here, we report a draft genome sequence of P. variotii no. 5; this draft was produced with the intent of investigating the mechanism of formaldehyde resistance. This is the first report of the genome sequence of any Paecilomyces species.
doi:10.1128/genomeA.01162-13
PMCID: PMC3886963  PMID: 24407650
5.  Purification and properties of S-hydroxymethylglutathione dehydrogenase of Paecilomyces variotii no. 5, a formaldehyde-degrading fungus 
AMB Express  2012;2:32.
S-hydroxymethylglutathione dehydrogenase from Paecilomyces variotii No. 5 strain (NBRC 109023), isolated as a formaldehyde-degrading fungus, was purified by a procedure that included ammonium sulfate precipitation, DEAE-Sepharose and hydroxyapatite chromatography and isoelectrofocusing. Approximately 122-fold purification was achieved with a yield of 10.5%. The enzyme preparation was homogeneous as judged by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 49 kDa by SDS-PAGE and gel filtration, suggesting that it is a monomer. Enzyme activity was optimal at pH 8.0 and was stable in the range of pH 7.0–10. The optimum temperature for activity was 40°C and the enzyme was stable up to 40°C. The isoelectric point was pH 5.8. Substrate specificity was very high for formaldehyde. Besides formaldehyde, the only aldehyde or alcohol tested that served as a substrate was pyruvaldehyde. Enzyme activity was enhanced by several divalent cations such as Mn2+ (179%), Ba2+ (132%), and Ca2+ (112%) but was completely inhibited by Ni2+, Fe3+, Hg2+, p-chloromercuribenzoate (PCMB) and cuprizone. Inactivation of the enzyme by sulfhydryl reagents (Hg2+ and PCMB) indicated that the sulfhydryl group of the enzyme is essential for catalytic activity.
doi:10.1186/2191-0855-2-32
PMCID: PMC3439253  PMID: 22731626
S-hydroxymethylglutathione dehydrogenase; Enzyme purification; Formaldehyde metabolism; Paecilomyces variotii; SH enzyme
6.  Putative Stress Sensors WscA and WscB Are Involved in Hypo-Osmotic and Acidic pH Stress Tolerance in Aspergillus nidulans ▿ † 
Eukaryotic Cell  2011;10(11):1504-1515.
Wsc proteins have been identified in fungi and are believed to be stress sensors in the cell wall integrity (CWI) signaling pathway. In this study, we characterized the sensor orthologs WscA and WscB in Aspergillus nidulans. Using hemagglutinin-tagged WscA and WscB, we showed both Wsc proteins to be N- and O-glycosylated and localized in the cell wall and membrane, implying that they are potential cell surface sensors. The wscA disruptant (ΔwscA) strain was characterized by reduced colony and conidia formation and a high frequency of swollen hyphae under hypo-osmotic conditions. The deficient phenotype of the ΔwscA strain was facilitated by acidification, but not by alkalization or antifungal agents. In contrast, osmotic stabilization restored the normal phenotype in the ΔwscA strain. A similar inhibition was observed in the wscB disruptant strain, but to a lesser extent. In addition, a double wscA and wscB disruptant (ΔwscA ΔwscB) strain was viable, but its growth was inhibited to a greater degree, indicating that the functions of the products of these genes are redundant. Transcription of α-1,3-glucan synthase genes (agsA and agsB) was significantly altered in the wscA disruptant strain, resulting in an increase in the amount of alkali-soluble cell wall glucan compared to that in the wild-type (wt) strain. An increase in mitogen-activated protein kinase (MpkA) phosphorylation was observed as a result of wsc disruption. Moreover, the transient transcriptional upregulation of the agsB gene via MpkA signaling was observed in the ΔwscA ΔwscB strain to the same degree as in the wt strain. These results indicate that A. nidulans Wsc proteins have a different sensing spectrum and downstream signaling pathway than those in the yeast Saccharomyces cerevisiae and that they play an important role in CWI under hypo-osmotic and acidic pH conditions.
doi:10.1128/EC.05080-11
PMCID: PMC3209062  PMID: 21926329
7.  Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans▿ †  
Eukaryotic Cell  2009;8(10):1465-1474.
Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.
doi:10.1128/EC.00371-08
PMCID: PMC2756868  PMID: 19648468

Results 1-7 (7)