A Novel Rapid Fungal Promoter Analysis System Using the Phosphopantetheinyl Transferase Gene, npgA, in Aspergillus nidulans

To develop a convenient promoter analysis system for fungi, a null-pigment mutant (NPG) of Aspergillus nidulans was used with the 4′-phosphopantetheinyl transferase (PPTase) gene, npgA, which restores the normal pigmentation in A. nidulans, as a new reporter gene. The functional organization of serially deleted promoter regions of the A. nidulans trpC gene and the Cryphonectria parasitica crp gene in filamentous fungi was representatively investigated to establish a novel fungal promoter assay system that depends on color complementation of the NPG mutant with the PPTase npgA gene. Several promoter regions of the trpC and crp genes were fused to the npgA gene containing the 1,034-bp open reading frame and the 966-bp 3’ downstream region from the TAA, and the constructed fusions were introduced into the NPG mutant in A. nidulans to evaluate color recovery due to the transcriptional activity of the sequence elements. Serial deletion of the trpC and crp promoter regions in this PPTase reporter assay system reaffirmed results in previous reports by using the fungal transformation step without a laborious verification process. This approach suggests a more rapid and convenient system than conventional analyses for fungal gene expression studies.

Overexpression and characterization of endo-cellulase A10 from Aspergillus nidulans in Pichia pastoris / 生物工程学报

Endo-cellulases are important to efficiently hydrolyze cellulose, and widely used in biotechnology. In this study, we overexpressed and characterized an endo-cellulase from Aspergillus nidulans. This endo-cellulase was successfully overexpressed in flasks and fermentor, and its concentration in fermentor reached 0.89 mg/mL. The optimal pH and temperature of the were 4.0 and 80 ℃ respectively, and it was very stable between pH 2.0 and 12.0. It was thermally stable below 60 ℃, whereas it was inactivated very quickly above 70 ℃. Its CMCase activity could be enhanced by Co²⁺, Mn²⁺ and Fe²⁺, whereas it was inhibited by Pb²⁺, Ni²⁺ and Cu²⁺. Therefore, this endo-cellulase exhibited good pH stability and thermostability below 60 ℃, and has the potential as commercial enzymes.

Morphoanatomic characterization of the stem and the leaf of Tabernaemontana catharinensis A. DC (Apocynaceae) and antimutagenic activity of its leaves / Caracterização morfoanatômica do caule e da folha de Tabernaemontana catharinensis A. DC (Apocynaceae) e atividade antimutagênica de suas folhas

ABSTRACT Tabernaemontana catharinensis A. DC (Apocynaceae) is used as a medicinal plant by the population. In order to contribute to the safe use of the plant as herbal medicine, this study aimed to morphoanatomically characterize the aereal vegetative organs of T. catharinensis and to evaluate the leaves’ mutagenic and antimutagenic activities. Histological blades of leaves and stem of T. catharinensis were performed; the methionine system (methG1) and Aspergillusnidulans conidia germination analysis were employed for mutagenic and antimutagenic evaluation. The morphoanatomic analysis did not show trichomes in the stem, petiole and leaf. Besides, it was observed both the presence of bi-collateral bundles - except in the foliar apex where the bundles were from the collateral type - as well as anamphistomatic leaf with paracyte stomata and sub-epidermal layer in the region of the leaf edges. The mutagenicity/antimutagenicity trial indicated a significant decrease of mutation frequency in comparison with the control group and showed that the T. catharinensis had antimutagenic activity within the type, time and form of treatment. Since the germination test showed that the conidia germination was accelerated from the bud phase, activities at the cell cycle level and polarized growth proved to be possible. The morphoanatomic analysis of the leaf and stem associated with the mutagenic and antimutagenic analyses contributes to the safe use of the plant by humans and also for the quality control of a possible phytotherapeutic drug.

RESUMO Tabernaemontana catharinensis A. DC (Apocynaceae) é utilizada como planta medicinal pela população. A fim de contribuir para o uso seguro da planta como medicinal, este trabalho teve como objetivo caracterizar morfoanatomicamente os órgãos vegetativos aéreos de T. catharinensis e avaliar a atividade mutagênica e antimutagênica de suas folhas. Foram realizados cortes histológicos da folha e do caule de T. catharinensis e, para a avaliação mutagênica e antimutagênica, foi utilizado o sistema metionina (methG1) e análise da germinação de conídios em Aspergillus nidulans. A análise morfoanatômica evidenciou a ausência de tricomas no caule, pecíolo e folha; presença de feixes bicolaterais, com exceção no ápice foliar cujos feixes são do tipo colateral; folha anfiestomática com estômatos paracíticos e camada subepidérmica na região do bordo foliar. O ensaio de mutagenicidade/antimutagenicidade mostrou uma diminuição significativa da frequência de mutação em relação ao controle, indicando que nesse tipo, tempo e forma de tratamento, T. catharinensis apresentou atividade antimutagênica. O ensaio de germinação evidenciou que houve aceleração da germinação dos conídios, a partir da fase de botão, indicando uma possível atuação em nível de ativação de ciclo celular e crescimento polarizado. A análise morfoanatômica da folha e do caule associados à análise mutagênica e antimutagênica, contribuem para o uso seguro da planta pela população e para o controle de qualidade de um possível fitoterápico.

Screening of Fungal Resources for the Production of Cellulases and Xylanases.

Aims: The final screening of fungal isolates aimed at applications based tertiary screening i.e. deinking of mixed office waste paper and saccharification of pearl millet stover and cellulases from selected fungal isolates were characterized. Study Design: An experimental study. Methodology: Samples from soil, compost and decaying wood were collected from different habitats and were screened based on growth over CMC-agar medium (primary screening), zone ratios and enzyme activities (secondary screening) and applications such as bio-deinking of mixed office paper and saccharification of pearl millet stover (tertiary screening). Results: 134 fungal isolates were selected during primary screening based on their growth. In secondary screening, fungal strains showing zone ratio of 3.0 or more were selected for application based tertiary screening. Two fungal isolates AKB-24 and AKB-25 were selected based on their applications in deinking of mixed office waste and saccharification of pearl millet stover after tertiary screening. Fungal isolates AKB-25 and AKB-24 were identified as Aspergillus nidulans and Penicillium sp. Optimum pH for FPase, endoglucanase, and glucosidase activities were 5.0 for both the fungal strains. Cellulases from A. nidulans AKB-25 were found moderately thermo-stable with optimum endoglucanase activity at 65ºC and optimal FPase and β-glucosidase activities at 60ºC. The maximal endoglucanase, FPase and β-glucosidase activities were observed at 55ºC for fungal strain Penicillium sp. AKB-24. Cellulases from both fungal strains were found stable up to 48 h at 50ºC. Conclusion: Aspergillus nidulans AKB-25 and Penicillium sp. AKB-24 were selected based on an extensive screening and enzymes from both fungal strains were found effective in bio-deinking of mixed office waste paper. Enzyme from Aspergillus nidulans AKB-25 was also found effective in saccharification of pearl millet stover.

Depletion of epsilon-COP in the COPI Vesicular Coat Reduces Cleistothecium Production in Aspergillus nidulans

We have previously isolated epsilon-COP, the alpha-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of epsilon-COP, the aneA+ gene for epsilon-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the epsilon-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of epsilon-COP did not rescue the thermo-sensitive growth defect of the alpha-COP mutant at 42degrees C. Together, these data show that epsilon-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

Role of LAMMER Kinase in Cell Wall Biogenesis during Vegetative Growth of Aspergillus nidulans

Depending on the acquisition of developmental competence, the expression of genes for beta-1,3-glucan synthase and chitin synthase was affected in different ways by Aspergillus nidulans LAMMER kinase. LAMMER kinase deletion, DeltalkhA, led to decrease in beta-1,3-glucan, but increase in chitin content. The DeltalkhA strain was also resistant to nikkomycin Z.

Avaliação da mutagenicidade de Piper methysticum L. f. no sistema methG1 em Aspergillus nidulans / Mutagenic evaluation of Piper methysticum L. f. in Aspergillus nidulans methG1 system

O extrato seco da raiz de Piper methysticum L. f. Forster (PIPERACEAE), a kava-kava, é usado no tratamento de diversos problemas envolvendo ansiedade como um dos sintomas. Por não causar dependência, sedação e ter ação ansiolítica, muitas pessoas têm recorrido a kava-kava para auxiliá-las no emagrecimento. Isto pode levar ao consumo indiscriminado da planta e acarretar riscos, pois todo medicamento fitoterápico deve respeitar limites de doses. Um risco na utilização de plantas medicinais é a toxicidade e, dentro deste, a mutagenicidade. Como a mutagenicidade está relacionada com a carcinogenicidade torna-se importante testar este potencial na kava-kava. Assim, o objetivo deste trabalho foi avaliar o potencial mutagênico do extrato seco da raiz de P. methysticum no sistema methG1 em Aspergillus nidulans. A linhagem utilizada foi a biA1methG1, auxotrófica para biotina e metionina. Conídios dormentes de colônias crescidas por cinco dias foram tratados com soluções da kava-kava nas concentrações de 0,35 mg mL-1 e 3,5 mg mL-1, e depois de 24h, semeados em meio seletivo contendo metionina, para análise dos sobreviventes, e sem metionina, para a análise dos mutantes. Os números de sobreviventes e mutantes dos tratamentos foram comparados aos do controle. Os resultados indicaram que o extrato da raiz da kava-kava é mutagênica, pois a freqüência de mutação dos tratamentos foi maior que da mutação espontânea, porém não ocorrendo diferença significativa entre as doses.

The dry root extract of Piper methysticum L. f. Forster (Piperaceae), kava-kava, is used as to treat several health problems involving anxiety symptoms. As it causes no addiction, it can be applied as a sedative and anxiolytic. Many people have been relying on kava-kava as an auxiliary treatment. This can lead to an indiscriminate plant consumption and lead to risks, because all phytotherapic medications must observe dosage limits. One risk in the folk medicinal plant use is toxicity, and within it, mutagenicity. As mutagenicity is closely related to carcinogenicity, it is important to test the kava-kava mutagenicity potential. Thus, the purpose of this work was to test the mutagenicity of the dry root extract of P. methysticum in the methG1 system of Aspergillus nidulans. The bia1methG1 lineage, which is auxotrophic for biotine and methione, was used. Conidia from five-day-old colonies were collected and treated with kava-kava solutions at 0.35 mg mL-1 and 3.5 mg mL-1 concentrations and, after 24h, they were planted in selective growth medium with and without methione, in order to analyze the survivors and mutants, respectively. The number of survivors and mutants analyzed under effect of the treatments was compared with the control. The results indicated that the kava-kava dry root extract is mutagenic, since the mutation frequency of the treatments was higher than spontaneous mutation, however, there were no differences between the doses tested.

pH Response Pathways in Fungi: Adapting to Host-derived and Environmental Signals

Microorganisms are significantly affected when the ambient pH of their environment changes. They must therefore be able to sense and respond to these changes in order to survive. Previous investigators have studied various fungal species to define conserved pH-responsive signaling pathways. One of these pathways, known as the Pal/Rim pathway, is activated in response to alkaline pH signals, ultimately targeting the PacC/Rim101 transcription factor. Although the central signaling components are conserved among divergent filamentous and yeast-like fungi, there is some degree of signaling specificity between fungal species. This specificity exists primarily in the downstream transcriptional targets of this pathway, likely allowing differential adaptation to species-specific environmental niches. In this review, the role of the Pal/Rim pathway in fungal pH response is discussed. Also highlighted are functional differences present in this pathway among human fungal pathogens, differences that allow these specialized microorganisms to survive in the various micro-environments of the infected human host.

Cytological characterization of an Aspergillus nidulans mutant from a strain with chromosomic duplication

A development mutant, named V103, was obtained spontaneously from the A strain of A. nidulans. The A strain contains a duplicated segment of chromosome I that has undergone translocation to chromosome II (I ¨ II). It is mitotically unstable and generates phenotypically deteriorated types, some with enhanced stability. The deteriorated variants of A. nidulans show abnormal development, exhibiting slower colony growth, variations in colony pigmentation and changes in conidiophore structure. The alterations observed in the conidiophore include fewer metulae and phialides, further elongation and ramification of these structures, delayed nuclear migration and the presence of secondary conidiophores.

Mechanism of sterigmatocystin biosynthesis regulation by pH in Aspergillus nidulans

External pH constitutes one of the most important environmental factors that control growth, metabolism and differentiation in microorganisms, including fungi. We have analyzed the effect of external pH on sterigmatocystin biosynthesis in Aspergillus nidulans. It was observed in repeated experiments that alkaline pH, in opposition to acid pH, increased sterigmatocystin production and the transcript levels of aflR, the master gene that regulates expression of the sterigmatocystin cluster in A. nidulans. It is known that pH effects in fungi operate mostly through the Pal/Pac signaling pathway, originally described in Aspergillus nidulans. Accordingly, we studied the role of this signaling pathway in ST biosynthesis. It was observed that aflR transcript levels were increased in the "alkalinity mimicking" mutant pacCc14 and were minimal in the "acidity mimicking" mutant palA1. No sterigmatocystin was produced by palA1 or pacC- mutants at neither acid or alkaline pH of incubation. Finally, fluG and flbA, genes known to regulate both conidiation and sterigmatocystin synthesis upstream in the regulatory cascade, were up-regulated at alkaline pH.

Induction of mitotic crossing-over in diploid strains of Aspergillus nidulansusing low-dose X-rays

As a contribution towards detecting the genetic effects of low doses of genotoxic physical agents, this paper deals with the consequences of low-dose X-rays in the Aspergillus nidulans genome. The irradiation doses studied were those commonly used in dental clinics (1-5 cGy). Even very low doses promoted increased mitotic crossing-over frequencies in diploid strains heterozygous for several genetic markers including the ones involved in DNA repair and recombination mechanisms. Genetic markers of several heterozygous strains were individually analyzed disclosing that some markers were especially sensitive to the treatments. These markers should be chosen as bio-indicators in the homozygotization index assay to better detect the recombinogenic/carcinogenic genomic effects of low-dose X-rays.

Evaluación tóxica y genotóxica del extracto fluido de Piper auritum H.B.K. / Toxic and genotoxic assessment of fluid extract from Piper auritum H.B.K.

Se evaluó la toxicidad aguda del extracto fluido de Piper auritum H.B.K. así como su actividad genotóxica a través de dos sistemas de ensayo a corto plazo, uno in vitro empleando la cepa D-30 de Aspergillus nidulans y otro in vivo, el ensayo de micronúcleos en médula ósea de ratón. Los resultados permiten concluir que el extracto fluido de P. auritum H.B.K. mostró muy baja toxicidad aguda y resultó genotóxico in vitro pero no in vivo .

The acute toxicity of fluid extract from Piper auritum H.B.K. as well as its genotoxic activity were evaluated through two short term assay systems, one in vitro using the D-30 strain of Aspergillus nidulans and, another in vivo, the micronuclei assay in mouse bone marrow. The results allow to conclude that the fluid extract from P.auritum H.B.K. showed a very low acute toxicity and that it proved to be genotoxic in vitro but not in vivo .

Genomic Organization of ancop Gene for alpha-COP Homolog from Aspergillus nidulans

We have cloned a alpha-COP homolog, ancop, from Aspergillus nidulans by colony hybridization of chromosome specific library using alpha-COP homologous fragment as a probe. The probe DNA was amplified with degenerated primers designed by comparison of conserved region of the amino acid sequences of Saccharomyces cerevisiae alpha-COP, Homo sapiens HEP-COP, and Drosophila melanogaster alpha-COP. Full length cDNA clone was also amplified by RT-PCR. Comparison of genomic DNA sequence with cDNA sequence obtained by RT-PCR revealed 7 introns. Amino acid sequence similarity search of the anCop with other alpha-COPs gave an overall identity of 52% with S. cerevisiae, 47% with human and bovine, 45% with Drosophila and Arabidopsis . In upstream region from the transcription start site, a putative TATA and CAAT motif were also identified.