EglD, a putative endoglucanase, with an expansin like domain is localized in the conidial cell wall of Aspergillus nidulans

Although the process of conidial germination in filamentous fungi has been extensively studied, many aspects remain to be elucidated since the asexual spore or conidium is vital in their life cycle. Breakage and reformation of cell wall polymer bonds along with the maintenance of cell wall plasticity during conidia germination depend upon a range of hydrolytic enzymes whose activity is analogous to that of expansins, a highly conserved group of plant cell wall proteins with characteristic wall loosening activity. In the current study, we identified and characterized the eglD gene in Aspergillus nidulans, an expansin-like gene the product of which shows strong similarities with bacterial and fungal endo-beta1,4-glucanases. However, we failed to show such activity in vitro. The eglD gene is constitutively expressed in all developmental stages and compartments of A. nidulans asexual life cycle. However, the EglD protein is exclusively present in conidial cell walls. The role of the EglD protein in morphogenesis, growth and germination rate of conidia was investigated. Our results show that EglD is a conidial cell wall localized expansin-like protein, which could be involved in cell wall remodeling during germination.     

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.     

The GanB Galpha-protein negatively regulates asexual sporulation and plays a positive role in conidial germination in Aspergillus nidulans

We isolated the ganB gene encoding the Galpha-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.     

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.     

Transformation of Aspergillus sp. and Trichoderma reesei using the pyrithiamine resistance gene (ptrA) of Aspergillus oryzae

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a beta-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.     

The role of the rodlet structure on the physicochemical properties of Aspergillus conidia

Physico-chemical properties of Aspergillus conidia rely on their outer cell-wall rodlet layer. In A. fumigatus and A. nidulans, the rodlet structure is due to an hydrophobin encoded by homologous rodA genes. To evaluate the role of the rodlet structure on the physico-chemical properties of conidia, we compared hydrophobicity, Lewis acid-base (i.e. electron donor/acceptor) characteristics and electrostatic charge of hydrophobin-less (rodletless) mutant and wild-type conidia of A. fumigatus and A. nidulans. The results obtained by aqueous-solvent partitioning assays, microsphere adhesion assays and microelectrophoresis showed that the disruption of the rodA gene modifies surface properties of A. fumigatus and A. nidulans conidia, and confirmed that the rodlet layer plays a key role in their physico-chemical behaviour. The absence of this layer on A. fumigatus spores led to the appearance of weakly basic and acidic characteristics, and had a slight effect on the hydrophobicity of conidia. Whereas in A. nidulans, it induced a basic character, a marked decrease in hydrophobicity and in the polarization capacity (electronegativity) of conidia. These physico-chemical differences between A. fumigatus and A. nidulans rodletless conidia may be attributed to differences in the composition of the conidial outer cell-wall of the two species.     

A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis

Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.     

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.     

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus.

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.     

Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans

Glutamine synthetase (GS), EC 6.3.1.2, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA. Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functional glnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic Anabaena glnA gene from the A. nidulans alcA promoter allowed full complementation of the A. nidulans glnADelta mutation. However, the A. nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A. nidulans glnA function when similarly expressed. Our studies with the glnADelta mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.     

Transformation with green fluorescent protein of Trichoderma harzianum 1051, a strain with biocontrol activity against Crinipellis perniciosa,the agent of witches'-broom disease of cocoa

A plasmid vector for fungal expression of an enhanced, red-shifted variant of the Aequoria victoriae green fluorescent protein was constructed by fusion of the EGFP gene to the highly expressed Aspergillus nidulans gpd promoter and the A. nidulans trpC terminator. This construction was introduced by cotransformation, using benomyl selection, into Trichoderma harzianum strain 1051, a strain being evaluated for the biological control of witches'-broom disease of cocoa caused by Crinipellis perniciosa. Epifluorescence microscopy was used to monitor germination and attachment of stable transformant conidia on the surface of C. perniciosa hyphae.     

Aluminum detoxification mechanism in Pseudomonas fluorescens is dependent on iron

Abstract Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant ( frA ). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans , while the fr A1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the fr A1 mutation. The absence of hexokinase activity in the fr A1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and l-arabinose catabolism. This suggests that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression.