Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.     

Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719.

The effect of the nitrogen source on the cellular activity of ferredoxin-nitrate reductase in different cyanobacteria was examined. In the unicellular species Anacystis nidulans, nitrate reductase was repressed in the presence of ammonium but de novo enzyme synthesis took place in media containing either nitrate or not nitrogen source, indicating that nitrate was not required as an obligate inducer. Nitrate reductase in A. nidulans was freed from ammonium repression by L-methionine-D,L-sulfoximine, an irreversible inhibitor of glutamine synthetase. Ammonium-promoted repression appears therefore to be indirect; ammonium has to be metabolized through glutamine synthetase to be effective in the repression of nitrate reductase. Unlike the situation in A. nidulans, nitrate appeared to play an active role in nitrate reductase synthesis in the filamentous nitrogen-fixing strains Anabaena sp. strain 7119 and Nostoc sp. strain 6719, with ammonium acting as an antagonist with regard to nitrate.     

应用实时荧光PCR技术检测构巢曲霉的初步研究

目的 根据构巢曲霉（Aspergillus nidulans）3-磷酸甘油醛脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase,GAPDH）基因特异位点设计并合成Taqman探针及引物,建立构巢曲霉实时荧光 PCR检测方法。方法 应用lasergene7.1软件对构巢曲霉与13种常见曲霉主要包括黑曲霉（A.niger）、烟曲霉（A.fumigatus）、杂色曲霉（A.versicolor）、土曲霉（A.terrus）、黄曲霉（A. flavus）、温特曲霉（A.wentii）、寄生曲霉（A. parasiticus）、泡盛曲霉（A.awamori）、米曲霉（A. oryzae ）、棒曲霉（A.cavatus）、赤曲霉（A.ruber ）、亮白曲霉（A.ochraceus）及赭曲霉（A.ochraceus）GAPDH基因序列比对分析,在特异位点设计引物和探针,建立构巢曲霉实时荧光 PCR检测方法,并对该方法进行特异性及敏感性分析。结果 用曲霉属22种41株不同曲霉及其他属的12株病原真菌验证实验表明,所建立的荧光PCR方法特异性强;检测灵敏度可达4.03×10－12μg/ml的模板DNA。 结论 应用实时荧光PCR技术能够有效检测构巢曲霉,该方法具有特异、灵敏、快速等特点,可在实际工作中应用。     

cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans

The role of cAMP signalling during germination of asexual spores (conidia) of the filamentous fungus Aspergillus nidulans was investigated. A. nidulans strains defective for adenylate cyclase (CyaA) or for the functionally overlapping cAMP-dependent protein kinase (PkaA) and newly characterized SchA protein kinase, homologous to Saccharomyces cerevisiae Sch9, show altered trehalose mobilization and kinetics of germ tube outgrowth, in addition to other defects in colony formation. cAMP-dependent trehalose breakdown is triggered by the addition of a carbon source independently of further catabolism, suggesting that cAMP signalling controls early events of conidial germination in response to carbon source sensing. Additional results suggest that cAMP has targets other than PkaA and SchA and that PkaA retains activity in the absence of cAMP. Conversely, PkaA regulates cAMP levels in A. nidulans because these are elevated by approximately 250-fold in a strain that lacks PkaA. Furthermore, analysis of mutant strains impaired in both adenylate cyclase and RasA GTPase previously implicated in the control of A. nidulans spore germination suggested that RasA and cAMP signalling proceed independently during germination in A. nidulans.     

有害疣孢霉菌与双孢蘑菇的互作关系

通过菌丝对峙、双重培养,以及对发病双孢蘑菇子实体的显微观察,探讨有害疣孢霉菌Mycogone perniciosa（MP0012）与双孢蘑菇Agaricus bisporus（As2796）之间的互作关系。结果表明,在菌丝对峙生长阶段有害疣孢霉菌菌丝不侵入双孢蘑菇菌丝体内,两者可交叉生长,对双孢蘑菇生长影响不显著;对峙与双重培养均显示有害疣孢霉菌菌丝会产生对双孢蘑菇菌丝生长的抑制作用的挥发性物质,造成双孢蘑菇菌丝扭结断裂。同时试验证实了双孢蘑菇菌丝会促进有害疣孢霉菌厚垣孢子的产生和萌发、菌丝生长和发育。侵染实验结果表明,有害疣孢霉菌可直接侵染双孢蘑菇子实体,引起双孢蘑菇子实体病害;对罹病子实体显微观察结果发现,发病前期双孢蘑菇子实体表面长出绒毛状病原菌丝,菌柄中空,菌褶褐变腐烂并长出病原菌丝;发病中期双孢蘑菇子实体内菌丝组织会出现萎缩裂解现象,在近有害疣孢霉菌菌丝一侧的双孢蘑菇子实体菌丝细胞壁被降解;发病后期双孢蘑菇子实体菌丝组织基本消失。由此初步判断有害疣孢霉菌对双孢蘑菇的寄生类型偏向于死体营养型。     

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.     

Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression

The fungal inhibitory effects of strain Lactobacillus plantarum MiLAB 393, producing broad-spectrum antifungal compounds, were evaluated. A co-cultivation method was set up to monitor effects on fungal growth and protein expression of growing Aspergillus nidulans with L. plantarum MiLAB 393. The effects of inhibitory metabolites produced by L. plantarum MiLAB 393, cyclo(l-Phe-l-Pro), lactic acid and 3-phenyllactic acid, were also investigated by addition of pure compounds to the growth medium of A. nidulans. The co-cultivation strongly affected the morphology of the fungal mycelium and decreased the biomass to 36% of control. Co-cultivation with Lactobacillus coryniformis MiLAB 123 gave only marginal morphological changes and minor biomass reduction, suggesting specific effects of L. plantarum MiLAB 393. The amount of several A. nidulans-proteins was increased during co-cultivation and by all of the inhibiting substances. This study shows that the growth of A. nidulans is inhibited during co-cultivation with L. plantarum MiLAB 393 and that the expression of fungal proteins is altered.     

RNA-mediated genetic transformation in Aspergillus nidulans

We developed a model-system for correcting genetic alterations of an Aspergillus nidulans strain (ribo, paba, bio, w, Acr) by treating protoplasts with total RNA extracted from another A. nidulans strain bearing wild type alleles for the same genetic markers. The results revealed the occurrence of a true genetic transformation. The phenomenon was RNA-dependent since it was abolished by pancreatic ribonuclease treatment. The term retrotransformation is proposed since the RNA messages artificially inserted into the mutant protoplast cells restore the wild type chromosomal information.     

Resolution of chromosomes III and VI of Aspergillus nidulans by pulsed-field gel electrophoresis shows that the penicillin biosynthetic pathway genes pcbAB, pcbC, and penDE are clustered on chromosome VI (3.0 megabases).

An improved electrophoretic molecular karyotype of Aspergillus nidulans ATCC 28901 has been obtained by contour-clamped electric field gel electrophoresis, which separates seven chromosomal bands and allows resolution of chromosomes III and VI. The three genes of the penicillin biosynthetic pathway, pcbAB, pcbC, and penDE, encoding alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase, and isopenicillin N acyltransferase, respectively, are clustered together on a chromosome of 3.0 Mg, corresponding to linkage group VI, whereas the argB gene was located on a chromosome of 3.4 Mb, corresponding to linkage group III. Three other strains of A. nidulans contained a modified chromosome III of about 3.1 Mb that overlaps with chromosome VI, forming a doublet. Resolution of chromosomes III and VI in strain ATCC 28901 allowed unequivocal mapping of the penicillin gene cluster on chromosome VI of A. nidulans.     

Aspergillus nidulans Cell Wall Composition and Function Change in Response to Hosting Several Aspergillus fumigatus UDP-Galactopyranose Mutase Activity Mutants

Deletion or repression of Aspergillus nidulans ugmA (AnugmA), involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a β-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA) crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA) strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK) substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63) was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased β-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced β-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and β-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.     

Transformation System for Aspergillus oryzae with Double Auxotrophic Mutations,niaD and sC

We developed a transformation system for Aspergillus oryzae using the Aspergillus nidulans sC gene encoding ATP sulfurylase as a selectable marker. The sC^? mutants can be readily isolated by positive selection for selenate resistance, thereby the niaD^? mutant strain of A. oryzae was bestowed with the sC^? mutation. Transformation of the A. oryzae host (niaD^?,sC^?) with the plasmid carrying A. nidulans sC gave random and multi-copy integrants, while that with the A. oryzae niaD-carrying plasmid occurred mainly by single-copy and homologous integration events (more than 50% frequency), indicating that with this transformation system, the transformation marker could be selected according to the integration pattern one desires.     

Vincristine induces somatic segregation,via mitotic crossing-over,in diploid cells of Aspergillus nidulans

Vincristine is an alkaloid widely used as an antineoplastic agent. In eukaryotic cells the drug causes blockage in the G2 phase of the cell cycle and an increase in the frequency of sister chromatid exchanges. Due to the fact that germinating Aspergillus nidulans cells spend most of their cycle in G2 phase, they provide an excellent system for the study of mitotic crossing-over. Taking into account that mitotic crossing-over occurs during G2 period, the evaluation of recombinagenic and aneugenic potential of vincristine is provided with regard to two diploid strains of A. nidulans: a wild strain (uvsH+//uvsH+) and a defective one in DNA repair (uvsH//uvsH). Drug toxicity and its effect on the asexual cycle of A. nidulans has been evaluated as well. Treatment of both strains with vincristine did not change colony growth in the culture, however cytological analyses showed aberrant conidiophores. Recombinagenic potential of vincristine was evaluated by induction of gene homozygosis originally present in heterozygosity diploid strains (Homozygotization Index). Results show that vincristine induces mitotic crossing-over and higher frequency of aneuploid mitotic segregants. The results also show the recombinagenic and aneuploidogenic potential of vincristine and suggest its participation in the induction of secondary malignancies.     

A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2.

Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria. In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A. nidulans. pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts. It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA. To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A. nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A. nidulans rRNA operon. This recombinant molecule was genetically and structurally stable during passage through A. nidulans and E. coli. The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A. nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.     

Isolation and functional analysis of a gene,tcsB, encoding a transmembrane hybrid-type histidine kinase from Aspergillus nidulans

We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (SLN1), which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an A. nidulans cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids. The predicted tcsB protein (TcsB) has two probable transmembrane regions in its N-terminal half and has a high degree of structural similarity to yeast Sln1p, a transmembrane hybrid-type histidine kinase. Overexpression of the tcsB cDNA suppressed the lethality of a temperature-sensitive osmosensing-defective sln1-ts yeast mutant. However, tcsB cDNAs in which the conserved phosphorylation site His(552) residue or the phosphorelay site Asp(989) residue had been replaced failed to complement the sln1-ts mutant. In addition, introduction of the tcsB cDNA into an sln1delta sho1delta yeast double mutant, which lacked two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an A. nidulans strain lacking the tcsB gene (tcsBdelta) and examined its phenotype. However, unexpectedly, the tcsBdelta strain did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media. Our results suggest that A. nidulans has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade.     

Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogenesis, and conidiation

Growing resistance to current anti-fungal drugs is spurring investigation of new targets, including those in fungal wall metabolism. Galactofuranose (Galf) is found in the cell walls of many fungi including Aspergillus fumigatus, which is currently the most prevalent opportunistic fungal pathogen in developed countries, and A. nidulans, a closely-related, tractable model system. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose into UDP-Galf prior to incorporation into the fungal wall. We deleted the single-copy UGM sequence (AN3112.4, which we call ugmA) from an A. nidulans nkuADelta strain, creating ugmADelta. Haploid ugmADelta strains were able to complete their asexual life cycle, showing that ugmA is not essential. However, ugmADelta strains had compact colonial growth, which was associated with substantially delayed and abnormal conidiation. Compared to a wildtype morphology strain, ugmADelta strains had aberrant hyphal morphology, producing wide, uneven, highly-branched hyphae, with thick, relatively electron-dense walls as visualized by transmission electron microscopy. These effects were partially remediated by growth on high osmolarity medium, or on medium containing 10 microg/mL Calcofluor, consistent with Galf being important in cell wall structure and/or function.     

The role of thiol species in the hypertolerance of Aspergillus sp. P37 to arsenic

Aspergillus sp. P37 is an arsenate-hypertolerant fungus isolated from a river in Spain with a long history of contamination with metals. This strain is able to grow in the presence of 0.2 M arsenate, i.e. 20-fold higher than the reference strain, Aspergillus nidulans TS1. Although Aspergillus sp. P37 reduces As(V) to As(III), which is slowly pumped out of the cell, the measured efflux of oxyanions is insufficient to explain the high tolerance levels of this strain. To gain an insight into this paradox, the accumulation of acid-soluble thiol species in Aspergillus sp. P37 when exposed to arsenic was compared with that of the arsenic-sensitive A. nidulans TS1 strain. Increasing levels of arsenic in the medium did not diminish the intracellular pool of reduced glutathione in Aspergillus sp. P37, in sharp contrast with the decline of glutathione in A. nidulans under the same conditions. Furthermore, concentrations of arsenic that were inhibitory for the sensitive A. nidulans strain (e.g. 50 mM and above) provoked a massive formation of vacuoles filled with thiol species. Because the major fraction of the cellular arsenic was present as the glutathione conjugate As(GS)3, it is plausible that the arsenic-hypertolerant phenotype of Aspergillus sp. P37 is in part due to an enhanced capacity to maintain a large intracellular glutathione pool under conditions of arsenic exposure and to sequester As(GS)3 in vacuoles. High pressure liquid chromatography analysis of cell extracts revealed that the contact of Aspergillus sp. P37 (but not A. nidulans) with high arsenic concentrations (> or =150 mM) induced the production of small quantities of a distinct thiol species indistinguishable from plant phytochelatin-2. Yet, we argue that phytochelatins do not explain arsenic resistance in Aspergillus, and we advocate the role of As(GS)3 complexes in arsenic detoxification.