RNA-dependent sterol aspartylation in fungi.

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes.

Square-plate culture method allows detection of differential gene expression and screening of novel, region-specific genes in Aspergillus oryzae.

When grown on solid agar medium, the mycelium of a filamentous fungus, Aspergillus oryzae, forms three morphologically distinct regions: the tip (T), white (W), and basal (B) regions. In this study, we developed the square-plate culture method, a novel culture method that enabled the extraction of mRNA samples from the three regions and analyzed the differential gene expression of the A. oryzae mycelium in concert with the microarray technique.

Isolation of Saccharomyces cerevisiae RNase T1 hypersensitive (rns) mutants and genetic analysis of the RNS1/DSL1 gene.

Overexpression of the rntA cDNA encoding RNase T1 derived from A. oryzae causes severe growth inhibition in S. cerevisiae.

Identification and characterization of rns4/vps32 mutation in the RNase T1 expression-sensitive strain of Saccharomyces cerevisiae: Evidence for altered ambient response resulting in transportation of the secretory protein to vacuoles.

We previously reported a genetic analysis of the growth-inhibitory effect caused by the overexpression of the Aspergillus oryzae rntA gene, encoding RNase T1 (Ribonuclease T1), in Saccharomyces cerevisiae. Subsequently, rns (ribonuclease T1 sensitive) mutants with mutations in the rns1 (DSL1), rns2 (UMP1), and rns3 (SEC17) genes, were identified. In the present study, rns4 (VPS32/SNF7) gene mutation was identified by complementation of tunicamycin sensitivity.

Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae.

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein.

Effects of protein transport inhibitors on the distribution and secretion of the fusion protein RntA-EGFP in Aspergillus oryzae.

The distribution of the secreted protein ribonuclease T1 (RntA) fused with the enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in hyphae of Aspergillus oryzae in the presence of a protein transport inhibitor, brefeldin A, cytochalasin A, or nocodazole. During treatment with the protein transport inhibitors, the distribution of RntA-EGFP changed and distinct patterns of fluorescence accumulation were observed. The addition of brefeldin A caused RntA-EGFP fluorescence to appear in reticular networks, and the disruption of the polymerization of actin filaments by cytochalasin A caused an increase in RntA-EGFP fluorescence intensity in the hyphae without accumulation in a specific cellular component.

Molecular cloning and functional characterization of avaB, a gene encoding Vam6p/Vps39p-like protein in Aspergillus nidulans.

It has been demonstrated that Saccharomyces cerevisiae Vam6p/Vps39p plays a critical role in the tethering steps of vacuolar membrane fusion by facilitating guanine nucleotide exchange on small guanosine triphosphatase (GTPase) Vam4p/Ypt7p. We report here the identification and characterization of a novel protein in Aspergillus nidulans, AvaB, that exhibits similarity to Vam6p/Vps39p and plays a critical role in vacuolar morphogenesis in A. nidulans.

Novel role of cytoplasmic dynein motor in maintenance of the nuclear number in conidia through organized conidiation in Aspergillus oryzae.

Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor protein complex. DhcA, cytoplasmic dynein heavy chain in Aspergillus oryzae, contained four P-loops involved in ATP binding which were conserved as in cytoplasmic dynein heavy chains of other organisms. The amino acid sequence of A.

In vivo visualization of the distribution of a secretory protein in Aspergillus oryzae hyphae using the RntA-EGFP fusion protein.

A fusion gene encoding ribonuclease T1-EGFP (rntA-egfp) was constructed and expressed to use it as a tool for studies on the secretory pathway in Aspergillus oryzae. The successful secretion of the intact RntA-EGFP fusion protein was detected by fluorescence measurement and Western analysis. With use of the RntA-EGFP system, we were able to see high fluorescence at hyphal tips and observe concentrated fluorescence at septa in basal cells during growth at optimal conditions.

n-Hexane sensitivity of Escherichia coli due to low expression of imp/ostA encoding an 87 kDa minor protein associated with the outer membrane.

Most Escherichia coli strains are resistant to n-hexane. E. coli OST4251 is a n-hexane-sensitive strain that was constructed by transferring the n-hexane-sensitive phenotype from a n-hexane-sensitive strain by P1 transduction.

Cloning and overexpression of beta-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide.

We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A.

Functional analysis of the calcineurin-encoding gene cnaA from Aspergillus oryzae: evidence for its putative role in stress adaptation.

The presence of putative STRE (stress response regulatory element) and HSF (heat-shock factor) transcription factor binding sites in the promoter region of the gene encoding calcineurin ( cnaA) from Aspergillus oryzae implicated a probable role for calcineurin in the stress response. The activity of calcineurin was enhanced during growth of the wild-type strain in the presence of 1 M NaCl (2.6-fold), at alkaline pH 10.

Molecular breeding of yeast with higher metal-adsorption capacity by expression of histidine-repeat insertion in the protein anchored to the cell wall.

A fusion protein of hexa-histidine repeat (His) and glycosylphosphatidylinositol (GPI)-anchor region of Saccharomyces cerevisiae Cwp1 with Aspergillus oryzae Taka-amylase A (TAA) was expressed on the yeast cell surface. The expressed fusion protein (TAA-His-Cwp1) was localized on the cell wall and demonstrated amylolytic activity. In comparison with the TAA-Cwp1 expressing strain, these cells exhibited 1.

Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae.

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1.

Visualization of vacuoles in Aspergillus oryzae by expression of CPY-EGFP.

Vacuolar carboxypeptidase Y (CPY) from Aspergillus nidulans was used to construct a CPY-EGFP fusion protein and expressed in A. oryzae to study vacuolar morphology and functions in A. oryzae.

Cloning and characterization of a gene (avaA) from Aspergillus nidulans encoding a small GTPase involved in vacuolar biogenesis.

Several genes that play roles in vacuolar biogenesis and targeting of proteins to vacuoles have been characterized in Saccharomyces cerevisiae. Although the vacuole is one of the most prominent compartments, little is known about molecular mechanism of vacuolar biogenesis in filamentous fungi. Vam4/Ypt7p, a small GTPase of the Rab/Ypt family in S.

Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis.

We isolated a strain from hop-resistant Lactobacillus hrevis ABBC45, which had lost a plasmid (pRH45) harboring a putative hop resistance gene, horA. The hop resistance level of this horA-deficient strain, named ABBC45(C), was initially low but gradually induced by repeated growth in media containing progressively increasing levels of hop compounds. Although the hop resistance level was substantially lower than that of the hop-adapted wild type strain, hop-adapted ABBC45(C) (ABBC45(CR)) was still capable of growing in beer, suggesting ABBC45 possesses at least two hop resistance mechanisms.

Cloning and characterization of vmaA, the gene encoding a 69-kDa catalytic subunit of the vacuolar H+-ATPase during alkaline pH mediated growth of Aspergillus oryzae.

Screening of a cDNA library constructed under alkaline pH mediated growth of Aspergillus oryzae implicated a vacuolar H+-ATPase gene (vmaA) as a putative candidate involved in alkaline pH adaptation. A. oryzae vmaA genomic DNA extended to 2072 bp including three introns and encoded a protein of 605 amino acids.

Observation of EGFP-visualized nuclei and distribution of vacuoles in Aspergillus oryzae arpA null mutant.

The arpA gene encoding Arp1 (actin-related protein) was previously cloned and characterized from Aspergillus oryzae. Phenotypes of the arpA null mutant indicate its requirement for normal nuclear distribution and morphology of conidiophores. In this study, we further characterized the function of the arpA gene in distribution of organelles.