Keon-Sang Chae

The veA gene activates sexual development in Aspergillus nidulans.

The previously isolated gene complementing the veA1 mutation was confirmed to be the veA gene. The determined nucleotide sequence of the gene demonstrated that there is an open reading frame (ORF) of a 573 amino acid polypeptide. The nucleotide sequence matched some clones of which functions were not assigned yet and the amino acid sequence matched that of Neurospora crassa VeA-related protein with 61% similarity. The nucleotide sequence of the veA1 mutant gene differed from that of the wild type gene by only one nucleotide and the nucleotide G in the initiation codon ATG of the VeA ORF was mutated to the nucleotide T. Then, the mutant ORF may use the 37th methionine codon of the wild type one as a new initiation codon. The veA transcript was present in the conidia and in mycelia cultured for up to 14h and expressed almost constitutively at an increased level throughout the asexual and sexual developmental processes, suggesting that it may act from a relatively early developmental stage. Null mutants of the gene never formed sexual structures, even under conditions where sexual development preferentially occurs in wild types. Over-expressors of the gene formed larger numbers of sexual structures with a much reduced number of conidial heads than a control strain (a veA1 mutant), even under conditions where wild type strains form little sexual structure but form conidial heads very well, such as in the presence of a salt at high concentration. Furthermore, over-expressors could form Hülle cells and cleistothecia, even in a liquid culture. These results indicated that the veA gene is a positive regulator in sexual development and simultaneously a negative one in asexual development.

Decolourization of azo dyes and a dye industry effluent by a white rot fungus Thelephora sp.

A white rot fungus Thelephora sp. was used for decolourization of azo dyes such as orange G (50 microM), congo red (50 microM), and amido black 10B (25 microM). Decolourization using the fungus was 33.3%, 97.1% and 98.8% for orange G, congo red and amido black 10B, respectively. An enzymatic dye decolourization study showed that a maximum of 19% orange G was removed by laccase at 15 U/ml whereas lignin peroxidase (LiP) and manganese dependent peroxidase (MnP) at the same concentration decolourized 13.5% and 10.8%, orange G, respectively. A maximum decolourization of 12.0% and 15.0% for congo red and amido black 10B, respectively, was recorded by laccase. A dye industry effluent was treated by the fungus in batch and continuous modes. A maximum decolourization of 61% was achieved on the third day in the batch mode and a maximum decolourization of 50% was obtained by the seventh day in the continuous mode. These results suggest that the batch mode of treatment using Thelephora sp. may be more effective than the continuous mode for colour removal from dye industry effluents.

The nsdD gene encodes a putative GATA‐type transcription factor necessary for sexual development of Aspergillus nidulans

The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomycete Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD gene has been isolated, and it is predicted to encode a GATA‐type transcription factor with the type IVb zinc finger DNA‐binding domain. The mRNA of the nsdD gene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and no nsdD mRNA was detected in conidia. Deletion of nsdD resulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating that nsdD is necessary for sexual development. In contrast, when the nsdD gene was over‐expressed, sexual‐specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that the nsdD gene functions in activating sexual development of A. nidulans. Multiple copies of the nsdD gene could suppress nsdB5 and veA1, indicating that either nsdD acts downstream of these genes or possibly functions in overlapping pathway(s).

The GanB Gα-Protein Negatively Regulates Asexual Sporulation and Plays a Positive Role in Conidial Germination in Aspergillus nidulans

We isolated the ganB gene encoding the Gα-protein homolog from Aspergillus nidulans. To investigate the cellular function of GanB, various mutant strains were isolated. Deletion of constitutively inactive ganB mutants showed conidiation and derepressed brlA expression in a submerged culture. Constitutive activation of GanB caused a reduction in hyphal growth and a severe defect in asexual sporulation. We therefore propose that GanB may negatively regulate asexual sporulation through the BrlA pathway. In addition, deletion or constitutive inactivation of GanB reduced germination rate while constitutive activation led to precocious germination. Furthermore, conidia of a constitutively active mutant could germinate even without carbon source. Taken together, these results indicated that GanB plays a positive role during germination, possibly through carbon source sensing, and negatively regulates asexual conidiation in A. nidulans.

Microbial decolorization of azo dyes and dye industry effluent by Fomes lividus

The white rot fungus, Fomes lividus, was isolated from the logs of Shorea robusta in the Western Ghats region of Tamil Nadu, India. The fungus was tested for decolorization of azo dyes such as orange G (50 μM) congo red (50 μM) amido black 10B (25 μM) and also for colour removal from dye industry effluents. The results revealed that the fungus could remove only 30.8% of orange G in the synthetic solution, whereas congo red and amido black 10B were removed by 74.0 and 98.9% respectively. A dye industry effluent was treated by the fungus in batch and continuous mode. In batch mode treatment, a maximum decolorization of 84.4% was achieved on day 4, and in continuous mode a maximum decolorization of 37.5% was obtained on day 5. The colour removal by the basidiomycete fungus might be due to adsorption of the dyes to the mycelial surface and metabolic breakdown. These results suggested that the batch mode treatment of Fomes lividus is one of the most efficient ways for colour removal in dye industry effluents.

The nsdC Gene Encoding a Putative C2H2-Type Transcription Factor Is a Key Activator of Sexual Development in Aspergillus nidulans

The formation of the Aspergillus nidulans fruiting body is affected by a number of genetic and environmental factors. Here, the nsdC (never in sexual development) gene—encoding a putative transcription factor carrying a novel type of zinc-finger DNA-binding domain consisting of two C2H2s and a C2HC motif that are highly conserved in most fungi but not in plants or animals—was investigated. Two distinct transcripts of 2.6 and 3.0 kb were generated from nsdC. The 2.6-kb mRNA accumulated differentially in various stages of growth and development, while the level of the 3.0-kb mRNA remained relatively constant throughout the life cycle. While the deletion of nsdC resulted in the complete loss of fruiting body formation under all conditions favoring sexual development, overexpression of nsdC not only enhanced formation of fruiting bodies (cleistothecia) but also overcame inhibitory effects of certain stresses on cleistothecial development, implying that NsdC is a key positive regulator of sexual development. Deletion of nsdC also retarded vegetative growth and hyperactive asexual sporulation, suggesting that NsdC is necessary not only for sexual development but also for regulating asexual sporulation negatively. Overexpression of veA or nsdD does not rescue the failure of fruiting body formation caused by nsdC deletion. Furthermore, nsdC expression is not affected by either VeA or NsdD, and vice versa, indicating that NsdC regulates sexual development independently of VeA or NsdD.

Membrane-bound methyltransferase complex VapA-VipC-VapB guides epigenetic control of fungal development.

Epigenetic and transcriptional control of gene expression must be coordinated in response to external signals to promote alternative multicellular developmental programs. The membrane-associated trimeric complex VapA-VipC-VapB controls a signal transduction pathway for fungal differentiation. The VipC-VapB methyltransferases are tethered to the membrane by the FYVE-like zinc finger protein VapA, allowing the nuclear VelB-VeA-LaeA complex to activate transcription for sexual development. Once the release from VapA is triggered, VipC-VapB is transported into the nucleus. VipC-VapB physically interacts with VeA and reduces its nuclear import and protein stability, thereby reducing the nuclear VelB-VeA-LaeA complex. Nuclear VapB methyltransferase diminishes the establishment of facultative heterochromatin by decreasing histone 3 lysine 9 trimethylation (H3K9me3). This favors activation of the regulatory genes brlA and abaA, which promote the asexual program. The VapA-VipC-VapB methyltransferase pathway combines control of nuclear import and stability of transcription factors with histone modification to foster appropriate differentiation responses.

Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene.

ABSTRACT We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.

Biological treatment of a pulp and paper industry effluent by Fomes lividus and Trametes versicolor

White rot fungi Fomes lividus and Trametes versicolor, isolated from the Western Ghats region of Tamil Nadu, India, were used to treat pulp and paper industry effluents on a laboratory scale and in a pilot scale. On the laboratory scale a maximum decolourization of 63.9% was achieved by T. versicolor on the fourth day. Inorganic chloride at a concentration of 765 mg/l, which corresponded to 227% of that in the untreated effluent, was liberated by F. lividus on the 10th day. The chemical oxygen demand (COD) was also reduced to 1984 mg/l (59.3%) by each of the two fungi. On the pilot scale, a maximum decolourization of 68% was obtained with the 6-day incubation by T. versicolor, inorganic chloride 475 mg/l (103%) was liberated on the seventh day by T. versicolor, and the COD was reduced to 1984 mg/l corresponding to 59.32% by F. lividus. These results suggested that F. lividus seems to be another candidate efficient for dechlorination of wastewater.

The MpkB MAP kinase plays a role in post-karyogamy processes as well as in hyphal anastomosis during sexual development in Aspergillus nidulans.

Two genes encoding MAP kinase homologs, designated as mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerate primers. Deletion and over-expression mutants of mpkC showed no detectable phenotypes under any external stress tested. Deletion of mpkB caused pleiotropic phenotypes including a failure in forming cleistothecia under any induction conditions for sexual development, increased Hülle cell production, slow hyphal growth and aberrant conidiophore morphology. Over-expression of mpkB led to increased cleistothecium production. While the transcripts of mpkB and mpkC were constitutively synthesized through the entire life cycle, their size and amount differed with developmental stages. An outcross test using fluorescent protein reporters showed that the mpkB deletion mutant could not form heterokaryons with wild type. Protoplast fusion experiments showed that the fusant of the mpkB mutant with wild type could undergo normal sexual development. However, heterokaryotic mycelia that were produced from a fusant between two mpkB deletion mutants could not form cleistothecia, although they did appear to form diploid nuclei. These results suggest that the MpkB MAP kinase is required for some post-karyogamy process as well as at the hyphal anastomosis stage to accomplish sexual development successfully.