Dataset Information

Functional analysis of the Aspergillus nidulans kinome.

ABSTRACT: The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs.

SUBMITTER: De Souza CP

PROVIDER: S-EPMC3591445 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Functional analysis of the Aspergillus nidulans kinome.

De Souza Colin P CP Hashmi Shahr B SB Osmani Aysha H AH Andrews Peter P Ringelberg Carol S CS Dunlap Jay C JC Osmani Stephen A SA

PloS one 20130307 3

The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have ...[more]

PMID: 23505451

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Project description:BACKGROUND: The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. RESULTS: In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. CONCLUSIONS: This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.

Project description:Transcription factors (TFs) with the basic leucin zipper domain are key elements of the stress response pathways in filamentous fungi. In this study, we functionally characterized the two bZIP type TFs AtfA and AtfB by deletion (Δ) and overexpression (OE) of their encoding genes in all combination: ΔatfA, ΔatfB, ΔatfAΔatfB, ΔatfAatfBOE, ΔatfBatfAOE, atfAOE, atfBOE and atfAOEatfBOE in Aspergillus nidulans. Based on our previous studies, ΔatfA increased the sensitivity of the fungus to oxidative stress mediated by menadione sodium bisulfite (MSB) and tert-butylhydroperoxide (tBOOH), while ΔatfB was not sensitive to any oxidative stress generating agents, namely MSB, tBOOH and diamide at all. Contrarily, the ΔatfB mutant was sensitive to NaCl, but tolerant to sorbitol. Overexpression of atfB was able to compensate the MSB sensitivity of the ΔatfA mutant. Heavy metal stress elicited by CdCl2 reduced diameter of the atfBOE and atfAOEatfBOE mutant colonies to about 50% of control colony, while the cell wall stress generating agent CongoRed increased the tolerance of the ΔatfA mutant. When we tested the heat stress sensitivity of the asexual spores (conidiospores) of the mutants, we found that conidiospores of ΔatfAatfBOE and ΔatfBatfAOE showed nearly 100% tolerance to heat stress. Asexual development was negatively affected by ΔatfA, while atfAOE and atfAOE coupled with ΔatfB increased the number of conidiospores of the fungus approximately 150% compared to the control. Overexpression of atfB led to a 25% reduction in the number of conidiospores, but increased levels of abaA mRNA and size of conidiospores. Sexual fruiting body (cleistothecium) formation was diminished in the ΔatfA and the ΔatfAΔatfB mutants, while relatively elevated in the ΔatfB and the ΔatfBatfAOE mutants. Production of the mycotoxin sterigmatocystin (ST) was decreased to undetectable levels in the ΔatfA mutant, yet ST production was restored in the ΔatfAΔatfB mutant, suggesting that ΔatfB can suppress ST production defect caused by ΔatfA. Levels of ST were also significantly decreased in the ΔatfAatfBOE, ΔatfBatfAOE and atfAOEatfBOE mutants.

Project description:BackgroundAspergillus nidulans is a member of a diverse group of filamentous fungi, sharing many of the properties of its close relatives with significance in the fields of medicine, agriculture and industry. Furthermore, A. nidulans has been a classical model organism for studies of development biology and gene regulation, and thus it has become one of the best-characterized filamentous fungi. It was the first Aspergillus species to have its genome sequenced, and automated gene prediction tools predicted 9,451 open reading frames (ORFs) in the genome, of which less than 10% were assigned a function.ResultsIn this work, we have manually assigned functions to 472 orphan genes in the metabolism of A. nidulans, by using a pathway-driven approach and by employing comparative genomics tools based on sequence similarity. The central metabolism of A. nidulans, as well as biosynthetic pathways of relevant secondary metabolites, was reconstructed based on detailed metabolic reconstructions available for A. niger and Saccharomyces cerevisiae, and information on the genetics, biochemistry and physiology of A. nidulans. Thereby, it was possible to identify metabolic functions without a gene associated, and to look for candidate ORFs in the genome of A. nidulans by comparing its sequence to sequences of well-characterized genes in other species encoding the function of interest. A classification system, based on defined criteria, was developed for evaluating and selecting the ORFs among the candidates, in an objective and systematic manner. The functional assignments served as a basis to develop a mathematical model, linking 666 genes (both previously and newly annotated) to metabolic roles. The model was used to simulate metabolic behavior and additionally to integrate, analyze and interpret large-scale gene expression data concerning a study on glucose repression, thereby providing a means of upgrading the information content of experimental data and getting further insight into this phenomenon in A. nidulans.ConclusionWe demonstrate how pathway modeling of A. nidulans can be used as an approach to improve the functional annotation of the genome of this organism. Furthermore we show how the metabolic model establishes functional links between genes, enabling the upgrade of the information content of transcriptome data.

Project description:Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulanssensu stricto clinical isolates. Just as in Aspergillus fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity.IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

Dataset Information

Functional analysis of the Aspergillus nidulans kinome.

Publications

Functional analysis of the Aspergillus nidulans kinome.

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