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. We report here the discovery of ergosteryl-3ß-O-l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3ß-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of "higher" fungi, including species of biotechnological interest, and, more importantly, in human pathogens like Aspergillus fumigatus We show that a bifunctional enzyme, ergosteryl-3ß-O-l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase-that produces aspartyl-tRNAAsp (Asp-tRNAAsp)-and of a Domain of Unknown Function 2156, which actually transfers aspartate from Asp-tRNAAsp onto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across "higher" fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity.

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. We previously reported that most S. cerevisiae mutant strains defective in translocation into the ER, ER-Golgi transport and vacuole formation exhibited hypersensitivity to expression of RNase T1. Screening for S. cerevisiae mutants that showed RNase T1 hypersensitivity resulted in the isolation of 38 (rns) mutant strains. Some of these mutants showed a variety of phenotypes including temperature-sensitive growth, hypersensitivity to G418, defect in invertase glycosylation and fragmented vacuoles. We identified the genes mutated in three of the rns mutants, rns1, rns2, and rns3, as DSL1, UMP1, and SEC17, respectively. Fluorescence microscopic observation showed that GFP or myc-tagged Rns1p was localized at the nuclear region in the cell. Two-hybrid screening revealed the interaction of Rns1p with a transcription factor Cin5p and a functionally unknown Ylr440cp. It was observed that HA-tagged Ylr440cp was localized to the ER and nuclear envelope.

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. cerevisiae, plays a vital role in homotypic vacuole fusion. We describe the isolation of the avaA gene from Aspergillus nidulans as a homologue of the VAM4/YPT7. Predicted 205 amino acids protein encoded by the avaA showed 67 and 72% identity with yeast Vam4/Ypt7p and human Rab7, respectively. The avaA disruptants exhibited highly fragmented vacuoles. We introduced mutations into the avaA gene, which alter nucleotide-binding characteristics of the gene products. Replacement of the avaA gene to the GDP-bound form mutant resulted in fragmentation of vacuoles. Overexpression of the GTP-bound form of avaA severely inhibited the hyphal growth and caused abnormal swelling of vacuoles. These results suggest that A. nidulans AvaA functions in the vacuolar biogenesis.

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. For further analysis of nuclear migration in living cells, an expression system consisting of a fusion protein of Aspergillus nidulans histone H2B and EGFP (H2B::EGFP) was used. This demonstrated diminished hyphal-tip growth rate and inefficient nuclear transport to apical regions in the arpA null mutant. Expression of H2B::EGFP also revealed an increase in the nuclear number of each conidium in the arpA null mutant, implicating a role for the arpA gene in controlling the nuclear movement into conidia. Furthermore, staining of vacuoles of the arpA null mutant with CMAC (cell tracker blue) suggested that the arpA gene is required for proper vacuolar distribution in addition to its role in normal nuclear distribution.

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. AvaB is comprised of 1058 amino acids with amino-terminal citron homology (CNH) and central clathrin homology (CLH) domains, as observed for other Vam6p/Vps39p family proteins. Disruption of avaB in A. nidulans resulted in the fragmentation of vacuoles and reduced growth rate under various growth conditions, implying its importance in maintaining vacuolar morphology and function. Yeast two-hybrid analysis demonstrated the interaction of AvaB with AvaA, a Vam4p/Ypt7p homolog in A. nidulans, as well as the homooligomer formation of AvaB, suggesting that AvaB performs its function through hetero- or homophilic protein-protein interactions.

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. VmaAp was homologous to Vma-1p from Neurospora crassa (71%), Vma1p from Saccharomyces cerevisiae (69%) and ATP6A2 from human (49%). The vmaA cDNA complemented S. cerevisiae V-ATPase disrupted strain (Deltavma1) was viable at alkaline pH 8.0 and in the presence of CaCl(2) (100 mM). Northern analysis revealed an enhanced expression of vmaA during growth of A. oryzae in alkaline medium (pH 10.0). The A. oryzae vmaA disruptant exhibited abnormally shrunken vacuoles and hyphal walls at pH 8.5 and a growth defect at pH 10.0, implicating an alkaline pH stress responsive role for vmaA in A. oryzae.

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. Hop resistance acquired by ABBC45(CR) gradually diminished to the pre-adapted level, when the strain was grown repeatedly in the absence of hop compounds. ABBC45(CR) was found to be cross-resistant to several structurally unrelated drugs, including ethidium bromide, daunomycin and nisin. In addition, ABBC45(CR) was shown to extrude ethidium in an energy-dependent manner, while ABBC45(C) did not show such activity. This indicates that the efflux pump was induced by adaptation to hop compounds. The efflux activity of ethidium was reduced by the addition of hop compounds, suggesting hop compounds are also the substrate of the efflux pump. It was also shown that the efflux activity was completely dissipated with the abolition of proton motive force (PMF). These results, taken together, suggest the hop resistance mechanism of ABBC45(C) is mediated by PMF-dependent multidrug efflux pump.

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. Expression of genes involved in protein synthesis was predominant in the T region; relative expression was, at most, six times higher in the T region compared to the other regions. Genes encoding hypothetical proteins were expressed at high levels in the W and B regions. In addition, genes coding transporters/permeases were predominantly transcribed in the B region. By analyzing the expression patterns of genes in the three regions, we demonstrated the dynamic changes in the regulation of gene expression that occur along the mycelium of filamentous fungi. Consequently, our study established a method to analyze and screen for region-specific genes whose function may be essential for morphogenesis and differentiation in filamentous fungi and whose traits may be beneficial to the biotechnology industry.

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. While the rns4 mutant exhibited sensitivity to ambient stress conditions (200 mM CaCl(2), 1M NaCl and pH 8.0), genome-wide expression analysis revealed a similar pattern of genes up-regulated as was observed under nitrogen depletion condition by Gasch et al. [Mol. Biol. Cell 11 (2000) 4241]. Notably, the genes participating in autophagy (ATG4 and ATG8), the genes encoding a vacuolar protease (PRB1), vacuolar protease inhibitors (PAI3, PBI2 and TFS1) and YHR138c (a PBI2 homolog) were up-regulated in the rns4 mutant. Interestingly, the RNase T1*-GFP fusion protein (*inactive form) expressed in the rns4 mutant strain localized at the ER and vacuole under both stress or no-stress conditions. In contrast, the RNase T1*-GFP fusion protein expressed in the wild-type strain could not be detected under no-stress conditions, however, a stress-dependent localization of the fusion protein was observed at the vacuole. Since, the rns4 mutant exhibited a partial starvation-like response in spite of a rich ambient environment, leading to transportation of the secretory protein to the vacuole and accumulation in the endoplasmic reticulum, the present findings implicate a novel role for Rns4/Vps32 in proper response and adaptation to ambient conditions.

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. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.

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. Cold or heat shock during growth caused the accumulation of EGFP fluorescence in vacuoles.

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. oryzae DhcA was similar to cytoplasmic dynein heavy chains from other organisms except for the N-terminus of Saccharomyces cerevisiae Dyn1. Disruption of dhcA gene in the region encoding four P-loop motifs resulted in a defective growth and perturbed distribution of nuclei and vacuoles. The dhcA disruptant exhibited an abnormal morphology of conidial heads and conidia with an increased nuclear number. The present study implicates a novel role of cytoplasmic dynein in maintenance of the nuclear number in conidia through an organized conidiation.

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. In contrast, RntA-EGFP fluorescence was distributed in different parts of a hypha during treatment with nocodazole, a compound that depolymerizes microtubules. In addition, quantitative analysis was performed using the RntA-EGFP visualization system to analyze the relative amount of RntA-EGFP secreted into the culture medium during treatment with the protein transport inhibitors.

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. While the fluorescence of EGFP was barely detectable in A. oryzae expressing CPY-EGFP grown under normal conditions at pH 5-6, the increase in pH of the growth medium towards alkalinity restored the fluorescence. In accordance with such an observation, the fluorescence of CPY-EGFP fusion protein in cell extract decreased in acidic pH condition, concomitant with lowered content of EGFP detected in A. oryzae grown under acidic pH conditions. The pH sensitivity of EGFP fluorescence and enhanced degradation of proteins in vacuoles under acidic pH conditions are thus proposed to result in the reduction of fluorescence in A. oryzae. Further, visualization of vacuoles revealed the presence of peculiar ring- or tube-like structures as distinct from normal spherical-shaped vacuoles.

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 1.6- to 2.8-fold higher adsorbing capacity for Cu(2+), Ni(2+), and Zn(2+).

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. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.

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. OST4251 is resistant to diphenyl ether, which is less harmful than n-hexane to micro-organisms. The genetic determinant responsible for this subtle difference in the solvent resistance is mapped at 1.2 min on the E. coli chromosome. Nucleotide sequence analysis showed that IS2 and IS5 had integrated upstream of the imp/ostA structural gene in OST4251. The integration of IS2 decreased the activity of the imp/ostA promoter. A product of the gene was identified immunologically as an 87 kDa minor protein associated with the outer membrane. Upon transformation with plasmids containing the imp/ostA gene, OST4251 produced a high level of the gene product in the membrane and acquired n-hexane resistance. Thus, the low level of promoter activity resulted in low Imp production and the n-hexane-sensitivity phenotype. It is likely that the gene product contributes to n-hexane resistance by reducing the influx of n-hexane.

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 10.0 (2.9-fold) and at 37 degrees C (1.6-fold). The induction of cnaA antisense expression resulted in reduced calcineurin activity (1.4-fold) and caused a growth defect under the stress conditions. Induction of a strain overexpressing cnaA resulted in an increase in calcineurin activity under stress conditions, such as the presence of 1 M NaCl (73%), alkaline pH 10.0 (70%), and a temperature of 37 degrees C (50%), in addition to tolerance to FK506 (a specific inhibitor of calcineurin). While a role for calcineurin in hyphal growth is well recognized, the present study suggests that stress adaptation mechanisms in filamentous fungi involve calmodulin/calcineurin-mediated signal transduction pathways.

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. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.