Publications by authors named "Claudio Scazzocchio"

65 Publications

In a Pair of Paralogous Isozymes Catalyze the First Committed Step of Leucine Biosynthesis in Either the Mitochondria or the Cytosol.

Front Microbiol 2020 4;11:1843. Epub 2020 Aug 4.

Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.

Divergence of paralogous pairs, resulting from gene duplication, plays an important role in the evolution of specialized or novel gene functions. Analysis of selected duplicated pairs has elucidated some of the mechanisms underlying the functional diversification of (. ) paralogous genes. Similar studies of the orthologous pairs extant in pre-whole genome duplication yeast species, such as () remain to be addressed. The genome of , an aerobic yeast, includes gene pairs generated by sporadic duplications. The genome of this organism comprises the and paralogous pair, annotated as putative α-isopropylmalate synthases (α-IPMSs), considered to be the orthologs of the / paralogous genes. The enzymes encoded by the latter two genes are mitochondrially located, differing in their sensitivity to leucine allosteric inhibition resulting in Leu4-Leu4 and Leu4-Leu9 sensitive dimers and Leu9-Leu9 relatively resistant homodimers. Previous work has shown that, in a Δ mutant, expression is increased and assembly of Leu9-Leu9 leucine resistant homodimers results in loss of feedback regulation of leucine biosynthesis, leading to leucine accumulation and decreased growth rate. Here we report that: (i) harbors a sporadic gene duplication, comprising the , syntenic with and , and the non-syntenic , arising from a pre-WGD event. (ii) That both, and encode leucine sensitive α-IPMSs isozymes, located in the mitochondria (Leu4) and the cytosol (Leu4BIS), respectively. (iii) That both, or complement the Δ Δ leucine auxotrophic phenotype and revert the enhanced transcription observed in a Δ mutant. The Δ growth mutant phenotype is only fully complemented when transformed with the syntenic mitochondrial isoform. and underwent a different diversification pathways than that leading to /. could be considered as the functional ortholog of , since its encoded isozyme can complement both the Δ Δ leucine auxotrophy and the Δ complex phenotype.
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http://dx.doi.org/10.3389/fmicb.2020.01843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418496PMC
August 2020

Complex intron generation in the yeast genus Lipomyces.

Sci Rep 2020 04 7;10(1):6022. Epub 2020 Apr 7.

Dept. of Biochemical Engineering, Faculty of Science and Technology, University of Debrecen, Debrecen, 4032, Hungary.

In primary transcripts of eukaryotic nuclear genes, coding sequences are often interrupted by U2-type introns. Such intervening sequences can constitute complex introns excised by consecutive splicing reactions. The origin of spliceosomal introns is a vexing problem. Sequence variation existent across fungal taxa provides means to study their structure and evolution. In one class of complex introns called [D] stwintrons, an (internal) U2 intron is nested within the 5'-donor element of another (external) U2 intron. In the gene for a reticulon-like protein in species of the ascomycete yeast genus Lipomyces, the most 5' terminal intron position is occupied by one of three complex intervening sequences consistent of differently nested U2 intron units, as demonstrated in L. lipofer, L. suomiensis, and L. starkeyi. In L. starkeyi, the donor elements of the constituent introns are abutting and the complex intervening sequence can be excised alternatively either with one standard splicing reaction or, as a [D] stwintron, by two consecutive reactions. Our work suggests how [D] stwintrons could emerge by the appearance of new functional splice sites within an extant intron. The stepwise stwintronisation mechanism may involve duplication of the functional intron donor element of the ancestor intron.
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http://dx.doi.org/10.1038/s41598-020-63239-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138796PMC
April 2020

A spliceosomal twin intron (stwintron) participates in both exon skipping and evolutionary exon loss.

Sci Rep 2019 07 9;9(1):9940. Epub 2019 Jul 9.

Department of Biochemical Engineering, University of Debrecen, Debrecen, 4032, Hungary.

Spliceosomal twin introns (stwintrons) are introns where any of the three consensus sequences involved in splicing is interrupted by another intron (internal intron). In Aspergillus nidulans, a donor-disrupted stwintron (intron-1) is extant in the transcript encoding a reticulon-like protein. The orthologous transcript of Aspergillus niger can be alternatively spliced; the exon downstream the stwintron could be skipped by excising a sequence that comprises this stwintron, the neighbouring intron-2, and the exon bounded by these. This process involves the use of alternative 3' splice sites for the internal intron, the resulting alternative intervening sequence being a longer 3'-extended stwintron. In 29 species of Onygenales, a multi-step splicing process occurs in the orthologous transcript, in which a complex intervening sequence including the stwintron and neigbouring intron-2, generates by three splicing reactions a "second order intron" which must then be excised with a fourth splicing event. The gene model in two species can be envisaged as one canonical intron (intron-1) evolved from this complex intervening sequence of nested canonical introns found elsewhere in Onygenales. Postulated splicing intermediates were experimentally verified in one or more species. This work illustrates a role of stwintrons in both alternative splicing and the evolution of intron structure.
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http://dx.doi.org/10.1038/s41598-019-46435-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616335PMC
July 2019

Reticulate evolution in eukaryotes: Origin and evolution of the nitrate assimilation pathway.

PLoS Genet 2019 02 21;15(2):e1007986. Epub 2019 Feb 21.

Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona, Catalonia, Spain.

Genes and genomes can evolve through interchanging genetic material, this leading to reticular evolutionary patterns. However, the importance of reticulate evolution in eukaryotes, and in particular of horizontal gene transfer (HGT), remains controversial. Given that metabolic pathways with taxonomically-patchy distributions can be indicative of HGT events, the eukaryotic nitrate assimilation pathway is an ideal object of investigation, as previous results revealed a patchy distribution and suggested that the nitrate assimilation cluster of dikaryotic fungi (Opisthokonta) could have been originated and transferred from a lineage leading to Oomycota (Stramenopiles). We studied the origin and evolution of this pathway through both multi-scale bioinformatic and experimental approaches. Our taxon-rich genomic screening shows that nitrate assimilation is present in more lineages than previously reported, although being restricted to autotrophs and osmotrophs. The phylogenies indicate a pervasive role of HGT, with three bacterial transfers contributing to the pathway origin, and at least seven well-supported transfers between eukaryotes. In particular, we propose a distinct and more complex HGT path between Opisthokonta and Stramenopiles than the one previously suggested, involving at least two transfers of a nitrate assimilation gene cluster. We also found that gene fusion played an essential role in this evolutionary history, underlying the origin of the canonical eukaryotic nitrate reductase, and of a chimeric nitrate reductase in Ichthyosporea (Opisthokonta). We show that the ichthyosporean pathway, including this novel nitrate reductase, is physiologically active and transcriptionally co-regulated, responding to different nitrogen sources; similarly to distant eukaryotes with independent HGT-acquisitions of the pathway. This indicates that this pattern of transcriptional control evolved convergently in eukaryotes, favoring the proper integration of the pathway in the metabolic landscape. Our results highlight the importance of reticulate evolution in eukaryotes, by showing the crucial contribution of HGT and gene fusion in the evolutionary history of the nitrate assimilation pathway.
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http://dx.doi.org/10.1371/journal.pgen.1007986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400420PMC
February 2019

Metabolic Gene Clusters in Eukaryotes.

Annu Rev Genet 2018 11 5;52:159-183. Epub 2018 Sep 5.

Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom; email:

In bacteria, more than half of the genes in the genome are organized in operons. In contrast, in eukaryotes, functionally related genes are usually dispersed across the genome. There are, however, numerous examples of functional clusters of nonhomologous genes for metabolic pathways in fungi and plants. Despite superficial similarities with operons (physical clustering, coordinate regulation), these clusters have not usually originated by horizontal gene transfer from bacteria, and (unlike operons) the genes are typically transcribed separately rather than as a single polycistronic message. This clustering phenomenon raises intriguing questions about the origins of clustered metabolic pathways in eukaryotes and the significance of clustering for pathway function. Here we review metabolic gene clusters from fungi and plants, highlight commonalities and differences, and consider how these clusters form and are regulated. We also identify opportunities for future research in the areas of large-scale genomics, synthetic biology, and experimental evolution.
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http://dx.doi.org/10.1146/annurev-genet-120417-031237DOI Listing
November 2018

A eukaryotic nicotinate-inducible gene cluster: convergent evolution in fungi and bacteria.

Open Biol 2017 12;7(12)

Department of Microbiology, University of Szeged Faculty of Science and Informatics, Szeged, Hungary (present address of ZH)

Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete , six loci, /AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/, AN11188/, AN11189/ and AN9177/, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the gene, constitutive mutations map in two discrete regions. is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of -heterocyclic compounds.
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http://dx.doi.org/10.1098/rsob.170199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746545PMC
December 2017

Emergence and loss of spliceosomal twin introns.

Fungal Biol Biotechnol 2017 6;4. Epub 2017 Oct 6.

Department of Biochemical Engineering, University of Debrecen, Debrecen, 4032 Hungary.

Background: In the primary transcript of nuclear genes, coding sequences-exons-usually alternate with non-coding sequences-introns. In the evolution of spliceosomal intron-exon structure, extant intron positions can be abandoned and new intron positions can be occupied. Spliceosomal twin introns ("stwintrons") are unconventional intervening sequences where a standard "internal" intron interrupts a canonical splicing motif of a second, "external" intron. The availability of genome sequences of more than a thousand species of fungi provides a unique opportunity to study spliceosomal intron evolution throughout a whole kingdom by means of molecular phylogenetics.

Results: A new stwintron was encountered in and . It is present across three classes of Leotiomyceta in the transcript of a well-conserved gene encoding a putative lipase (). It occupies the same position as a standard intron in the orthologue gene in species of the early divergent classes of the Pezizomycetes and the Orbiliomycetes, suggesting that an internal intron has appeared within a pre-extant intron. On the other hand, the stwintron has been lost from certain taxa in Leotiomycetes and Eurotiomycetes at several occasions, most likely by a mechanism involving reverse transcription and homologous recombination. Another ancient stwintron present across whole Pezizomycotina orders-in the transcript of the bifunctional biotin biosynthesis gene -occurs at the same position as a standard intron in many species of non-Dikarya. Nevertheless, also the stwintron has disappeared from certain lineages within the taxa where it occurs, i.e., Sordariomycetes and Botryosphaeriales. Intriguingly, only the internal intron was lost from the Sordariomycetes stwintron at all but one occasion, leaving a standard intron in the same position, while where the putative lipase stwintron was lost, no intronic sequences remain.

Conclusions: Molecular phylogeny of the peptide product was used to monitor the existence and fate of a stwintron in the transcripts of two neatly defined fungal genes, encoding well conserved proteins. Both defining events-stwintron emergence and loss-can be explained with extant models for intron insertion and loss. We thus demonstrate that stwintrons can serve as model systems to study spliceosomal intron evolution.
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http://dx.doi.org/10.1186/s40694-017-0037-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639578PMC
October 2017

A mechanism for a single nucleotide intron shift.

Nucleic Acids Res 2017 Sep;45(15):9085-9092

Department of Biochemical Engineering, University of Debrecen, 4032, Hungary.

Spliceosomal introns can occupy nearby rather than identical positions in orthologous genes (intron sliding or shifting). Stwintrons are complex intervening sequences, where an 'internal' intron interrupts one of the sequences essential for splicing, generating after its excision, a newly formed canonical intron defined as 'external'. In one experimentally demonstrated configuration, two alternatively excised internal introns, overlapping by one G, disrupt respectively the donor and the acceptor sequence of an external intron, leading to mRNAs encoding identical proteins. In a gene encoding a DHA1 antiporter in Pezizomycotina, we find a variety of predicted intron configurations interrupting the DNA stretch encoding a conserved peptidic sequence. Some sport a stwintron where the internal intron interrupts the donor of the external intron (experimentally confirmed for Aspergillus nidulans). In others, we found and demonstrate (for Trichoderma reesei) alternative, overlapping internal introns. Discordant canonical introns, one nt apart, are present in yet other species, exactly as predicted by the alternative loss of either of the internal introns at the DNA level from an alternatively spliced stwintron. An evolutionary pathway of 1 nt intron shift, involving an alternatively spliced stwintron intermediate is proposed on the basis of the experimental and genomic data presented.
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http://dx.doi.org/10.1093/nar/gkx520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587772PMC
September 2017

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

Authors:
Ronald P de Vries Robert Riley Ad Wiebenga Guillermo Aguilar-Osorio Sotiris Amillis Cristiane Akemi Uchima Gregor Anderluh Mojtaba Asadollahi Marion Askin Kerrie Barry Evy Battaglia Özgür Bayram Tiziano Benocci Susanna A Braus-Stromeyer Camila Caldana David Cánovas Gustavo C Cerqueira Fusheng Chen Wanping Chen Cindy Choi Alicia Clum Renato Augusto Corrêa Dos Santos André Ricardo de Lima Damásio George Diallinas Tamás Emri Erzsébet Fekete Michel Flipphi Susanne Freyberg Antonia Gallo Christos Gournas Rob Habgood Matthieu Hainaut María Laura Harispe Bernard Henrissat Kristiina S Hildén Ryan Hope Abeer Hossain Eugenia Karabika Levente Karaffa Zsolt Karányi Nada Kraševec Alan Kuo Harald Kusch Kurt LaButti Ellen L Lagendijk Alla Lapidus Anthony Levasseur Erika Lindquist Anna Lipzen Antonio F Logrieco Andrew MacCabe Miia R Mäkelä Iran Malavazi Petter Melin Vera Meyer Natalia Mielnichuk Márton Miskei Ákos P Molnár Giuseppina Mulé Chew Yee Ngan Margarita Orejas Erzsébet Orosz Jean Paul Ouedraogo Karin M Overkamp Hee-Soo Park Giancarlo Perrone Francois Piumi Peter J Punt Arthur F J Ram Ana Ramón Stefan Rauscher Eric Record Diego Mauricio Riaño-Pachón Vincent Robert Julian Röhrig Roberto Ruller Asaf Salamov Nadhira S Salih Rob A Samson Erzsébet Sándor Manuel Sanguinetti Tabea Schütze Kristina Sepčić Ekaterina Shelest Gavin Sherlock Vicky Sophianopoulou Fabio M Squina Hui Sun Antonia Susca Richard B Todd Adrian Tsang Shiela E Unkles Nathalie van de Wiele Diana van Rossen-Uffink Juliana Velasco de Castro Oliveira Tammi C Vesth Jaap Visser Jae-Hyuk Yu Miaomiao Zhou Mikael R Andersen David B Archer Scott E Baker Isabelle Benoit Axel A Brakhage Gerhard H Braus Reinhard Fischer Jens C Frisvad Gustavo H Goldman Jos Houbraken Berl Oakley István Pócsi Claudio Scazzocchio Bernhard Seiboth Patricia A vanKuyk Jennifer Wortman Paul S Dyer Igor V Grigoriev

Genome Biol 2017 02 14;18(1):28. Epub 2017 Feb 14.

US Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, 94598, USA.

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.

Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli.

Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.
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http://dx.doi.org/10.1186/s13059-017-1151-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5307856PMC
February 2017

Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

Fungal Genet Biol 2015 Dec 26;85:7-13. Epub 2015 Oct 26.

Dept. of Biochemical Engineering, Faculty of Science and Technology, University of Debrecen, H-4032 Debrecen, Hungary.

Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.
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http://dx.doi.org/10.1016/j.fgb.2015.10.004DOI Listing
December 2015

Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha.

PLoS One 2015 3;10(9):e0135416. Epub 2015 Sep 3.

Fungal Genetics and Genomics Unit, Division of Microbial Genetics and Pathogen Interactions, BOKU-University of Natural Resources and Life Sciences Vienna, University and Research Center Tulln, Konrad Lorenz Strasse 24, 3430, Tulln/Donau, Austria; Health and Environment Department, Austrian Institute of Technology GmbH (AIT), University and Research Center Tulln, Konrad Lorenz Strasse 24, 3430, Tulln/Donau, Austria.

A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135416PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559421PMC
May 2016

Refining the pH response in Aspergillus nidulans: a modulatory triad involving PacX, a novel zinc binuclear cluster protein.

Mol Microbiol 2015 Dec 16;98(6):1051-72. Epub 2015 Oct 16.

Section of Microbiology, Imperial College London, Flowers Building, Armstrong Road, London, SW7 2AZ, UK.

The Aspergillus nidulans PacC transcription factor mediates gene regulation in response to alkaline ambient pH which, signalled by the Pal pathway, results in the processing of PacC(72) to PacC(27) via PacC(53). Here we investigate two levels at which the pH regulatory system is transcriptionally moderated by pH and identify and characterise a new component of the pH regulatory machinery, PacX. Transcript level analysis and overexpression studies demonstrate that repression of acid-expressed palF, specifying the Pal pathway arrestin, probably by PacC(27) and/or PacC(53), prevents an escalating alkaline pH response. Transcript analyses using a reporter and constitutively expressed pacC trans-alleles show that pacC preferential alkaline-expression results from derepression by depletion of the acid-prevalent PacC(72) form. We additionally show that pacC repression requires PacX. pacX mutations suppress PacC processing recalcitrant mutations, in part, through derepressed PacC levels resulting in traces of PacC(27) formed by pH-independent proteolysis. pacX was cloned by impala transposon mutagenesis. PacX, with homologues within the Leotiomyceta, has an unusual structure with an amino-terminal coiled-coil and a carboxy-terminal zinc binuclear cluster. pacX mutations indicate the importance of these regions. One mutation, an unprecedented finding in A. nidulans genetics, resulted from an insertion of an endogenous Fot1-like transposon.
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http://dx.doi.org/10.1111/mmi.13173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832277PMC
December 2015

Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

PLoS Genet 2015 Jul 1;11(7):e1005297. Epub 2015 Jul 1.

Fungal Genetics and Genomics Unit, Department of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Life Science, Vienna, Vienna, Austria; Health and Environment Department, Austrian Institute of Technology GmbH-AIT, University and Research Center Tulln, Tulln an der Donau, Austria.

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol.
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http://dx.doi.org/10.1371/journal.pgen.1005297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488483PMC
July 2015

Minos as a novel Tc1/mariner-type transposable element for functional genomic analysis in Aspergillus nidulans.

Fungal Genet Biol 2015 Aug 25;81:1-11. Epub 2015 May 25.

Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15784, Greece. Electronic address:

Transposons constitute powerful genetic tools for gene inactivation, exon or promoter trapping and genome analyses. The Minos element from Drosophila hydei, a Tc1/mariner-like transposon, has proved as a very efficient tool for heterologous transposition in several metazoa. In filamentous fungi, only a handful of fungal-specific transposable elements have been exploited as genetic tools, with the impala Tc1/mariner element from Fusarium oxysporum being the most successful. Here, we developed a two-component transposition system to manipulate Minos transposition in Aspergillus nidulans (AnMinos). Our system allows direct selection of transposition events based on re-activation of niaD, a gene necessary for growth on nitrate as a nitrogen source. On average, among 10(8) conidiospores, we obtain up to ∼0.8×10(2) transposition events leading to the expected revertant phenotype (niaD(+)), while ∼16% of excision events lead to AnMinos loss. Characterized excision footprints consisted of the four terminal bases of the transposon flanked by the TA target duplication and led to no major DNA rearrangements. AnMinos transposition depends on the presence of its homologous transposase. Its frequency was not significantly affected by temperature, UV irradiation or the transcription status of the original integration locus (niaD). Importantly, transposition is dependent on nkuA, encoding an enzyme essential for non-homologous end joining of DNA in double-strand break repair. AnMinos proved to be an efficient tool for functional analysis as it seems to transpose in different genomic loci positions in all chromosomes, including a high proportion of integration events within or close to genes. We have used Minos to obtain morphological and toxic analogue resistant mutants. Interestingly, among morphological mutants some seem to be due to Minos-elicited over-expression of specific genes, rather than gene inactivation.
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http://dx.doi.org/10.1016/j.fgb.2015.05.007DOI Listing
August 2015

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

Eukaryot Cell 2015 Jun 3;14(6):564-77. Epub 2015 Apr 3.

Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.
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http://dx.doi.org/10.1128/EC.00033-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452578PMC
June 2015

Origin, diversification and substrate specificity in the family of NCS1/FUR transporters.

Mol Microbiol 2015 Jun 7;96(5):927-50. Epub 2015 Apr 7.

Faculty of Biology, University of Athens, Panepistimioupolis, Athens, 15784, Greece.

NCS1 proteins are H(+)/Na(+) symporters specific for the uptake of purines, pyrimidines and related metabolites. In this article, we study the origin, diversification and substrate specificity of fungal NCS1 transporters. We show that the two fungal NCS1 sub-families, Fur and Fcy, and plant homologues originate through independent horizontal transfers from prokaryotes and that expansion by gene duplication led to the functional diversification of fungal NCS1. We characterised all Fur proteins of the model fungus Aspergillus nidulans and discovered novel functions and specificities. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. Most importantly, we predict and confirm that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward-facing selective gate. Our evolutionary and structure-function analysis contributes in the understanding of the molecular mechanisms underlying the functional diversification of eukaryotic NCS1 transporters, and in particular, forward the concept that selective channel-like gates might contribute to substrate specificity.
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http://dx.doi.org/10.1111/mmi.12982DOI Listing
June 2015

Functional characterization of NAT/NCS2 proteins of Aspergillus brasiliensis reveals a genuine xanthine-uric acid transporter and an intrinsically misfolded polypeptide.

Fungal Genet Biol 2015 Feb 29;75:56-63. Epub 2015 Jan 29.

Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15784, Greece. Electronic address:

The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterized NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterized mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high-capacity, H(+)/xanthine-uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed.
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http://dx.doi.org/10.1016/j.fgb.2015.01.009DOI Listing
February 2015

Fungal biology in the post-genomic era.

Fungal Biol Biotechnol 2014 14;1. Epub 2014 Oct 14.

Department of Microbiology, Imperial College, London, SW7 2AZ UK.

In this review I give a personal perspective of how fungal biology has changed since I started my Ph. D. in 1963. At that time we were working in the shadow of the birth of molecular biology as an autonomous and reductionistic discipline, embodied in Crick's central dogma. This first period was methodologically characterised by the fact that we knew what genes were, but we could not access them directly. This radically changed in the 70s-80s when gene cloning, reverse genetics and DNA sequencing become possible. The "next generation" sequencing techniques have produced a further qualitative revolutionary change. The ready access to genomes and transcriptomes of any microbial organism allows old questions to be asked in a radically different way and new questions to be approached. I provide examples chosen somewhat arbitrarily to illustrate some of these changes, from applied aspects to fundamental problems such as the origin of fungal specific genes, the evolutionary history of genes clusters and the realisation of the pervasiveness of horizontal transmission. Finally, I address how the ready availability of genomes and transcriptomes could change the status of model organisms.
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http://dx.doi.org/10.1186/s40694-014-0007-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611559PMC
October 2014

A dually located multi-HMG-box protein of Aspergillus nidulans has a crucial role in conidial and ascospore germination.

Mol Microbiol 2014 Oct 11;94(2):383-402. Epub 2014 Sep 11.

University of Szeged Faculty of Sciences and Informatics, Department of Microbiology, H-6726, Szeged, Közép fasor 52, Hungary.

Seven HMG-box proteins of Aspergillus nidulans have been identified in the genomic databases. Three of these have the characteristics of non-specific DNA-binding proteins. One of these, AN1267 (HmbB), comprises one canonical HMG-box in its C-terminus and upstream of the canonical box two structurally related boxes, to be called Shadow-HMG-boxes. This protein defines, together with the Podospora anserina mtHMG1, a clade of proteins present in the Pezizomycotina, with orthologues in some of the Taphrinomycotina. HmbB localizes primarily to the mitochondria but occasionally in nuclei. The deletion of the cognate gene results in a number of pleiotropic effects, including those on hyphal morphology, sensitivity to oxidative stress, absence of sterigmatocystin production and changes in the profile of conidial metabolites. The most striking phenotype of deletion strains is a dramatic decrease in conidial and ascospore viability. We show that this is most likely due to the protein being essential to maintain mitochondrial DNA in spores.
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http://dx.doi.org/10.1111/mmi.12772DOI Listing
October 2014

Purine utilization proteins in the Eurotiales: cellular compartmentalization, phylogenetic conservation and divergence.

Fungal Genet Biol 2014 Aug 24;69:96-108. Epub 2014 Jun 24.

Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15784, Greece. Electronic address:

The purine utilization pathway has been thoroughly characterized in Aspergillus nidulans. We establish here the subcellular distribution of seven key intracellular enzymes, xanthine dehydrogenase (HxA), urate oxidase (UaZ), 5-hydroxy-isourate hydrolase (UaX), 2-oxo-4-hydroxy-4-carboxy ureido imidazoline decarboxylase (UaW), allantoinase (AlX), allantoicase (AaX), ureidoglycolate lyase (UglA), and the fungal-specific α-ketoglutarate Fe(II)-dependent dioxygenase (XanA). HxA, AlX, AaX, UaW and XanA are cytosolic, while UaZ, UaX and UglA are peroxisomal. Peroxisomal localization was confirmed by using appropriate pex mutants. The pathway is largely, but not completely conserved in the Eurotiomycetes, noticeably in some species AaX is substituted by an alternative enzyme of probable bacterial origin. UaZ and the urate-xanthine UapA and UapC transporters, are also localized in specific cells of the conidiophore. We show that metabolic accumulation of uric acid occurring in uaZ null mutations is associated with an increased frequency of appearance of morphologically distinct colony sectors, diminished conidiospore production, UV resistance and an altered response to oxidation stress, which may provide a rationale for the conidiophore-specific localization. The pathway-specific transcription factor UaY is localized in both the cytoplasm and nuclei under non-inducing conditions, but it rapidly accumulates exclusively to the nuclei upon induction by uric acid.
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http://dx.doi.org/10.1016/j.fgb.2014.06.005DOI Listing
August 2014

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H⁺ transporters in fungi and plants.

Open Biol 2014 Jun;4(6):140070

Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

We present the first account of the structure-function relationships of a protein of the subfamily of urea/H(+) membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.
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http://dx.doi.org/10.1098/rsob.140070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077062PMC
June 2014

In vitro and in silico analysis of the Aspergillus nidulans DNA-CreA repressor interactions.

J Biomol Struct Dyn 2014 Dec 15;32(12):2033-41. Epub 2013 Oct 15.

a Biologia Molecular Departamento de Bioquímica Clínica, Facultad de Química , UdelaR , General Flores 2124, C.P. 1157, 11800 Montevideo , Uruguay .

The CreA protein mediates carbon catabolite repression in the fungus Aspergillus nidulans. Its DNA-binding domain belongs to the Cys2-His2 class, binding specifically to a 5' SYGGRG 3' nucleotide sequence. EMSA experiments showed that the CreA(G27D) mutation resulted in a 30-fold increase of the Kdiss, and footprinting revealed a altered pattern of protein/DNA contacts. We modeled the CreA and the CreA(G27D) complexes in silico. A 15 ns molecular dynamics simulation of the solvated CreA(G27D) and CreA models was carried out using the MOE 2007.09 suite and the Amber99 force field. We have focused our analysis in residues Arg14, Glu16, His17, and Arg20 and Arg44, Asp46, and Arg50, previously, shown to be responsible for the specific contacts of the two Zn fingers. The electrostatic and the total potential energies showed the CreA(G27D) mutation to decrease the affinity of the complex, in agreement with the Kdiss's values. The in silico approach highlighted the role of the inter-finger linker. We identified several differential structural characteristics of the CreA and CreA(G27D)/DNA complexes and observed that the latter resulted in a lower dynamic flexibility of the complex.
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http://dx.doi.org/10.1080/07391102.2013.843474DOI Listing
December 2014

In praise of erroneous hypotheses.

Fungal Genet Biol 2013 Sep-Oct;58-59:126-31. Epub 2013 Aug 20.

Department of Microbiology, Imperial College, London SW7 2AZ, United Kingdom; Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris-Sud, 91405 Orsay, France. Electronic address:

In the sixties Cove and Pateman discovered that mutants of Aspergillus nidulans lacking nitrate reductase activity were constitutive for the expression of genes induced by nitrate and dependent on the transcription factor NirA. They proposed that the nitrate protein acted as a repressor, preventing the transcription factor activity of NirA. Nitrate-mediated regulation behaved similarly in other organisms. This "autogenous regulation hypothesis" has recently shown to be erroneous, in the very organism for which it was first proposed. Nevertheless this erroneous hypothesis have led to a thorough dissection of the process of regulation of nitrate assimilation and more importantly to a hypothesis bearing on the origin of metabolite-responsive transcription factors. In this article I discuss the heuristic value and evolutionary importance of autogenous regulation.
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http://dx.doi.org/10.1016/j.fgb.2013.08.008DOI Listing
April 2014

Spliceosome twin introns in fungal nuclear transcripts.

Fungal Genet Biol 2013 Aug 19;57:48-57. Epub 2013 Jun 19.

Department of Biochemical Engineering, Faculty of Science and Technology, University of Debrecen, 4010 Debrecen, Hungary.

The spliceosome is an RNA/protein complex, responsible for intron excision from eukaryotic nuclear transcripts. In bacteria, mitochondria and plastids, intron excision does not involve the spliceosome, but occurs through mechanisms dependent on intron RNA secondary and tertiary structure. For group II/III chloroplast introns, "twintrons" (introns within introns) have been described. The excision of the external intron, and thus proper RNA maturation, necessitates prior removal of the internal intron, which interrupts crucial sequences of the former. We have here predicted analogous instances of spliceosomal twintrons ("stwintrons") in filamentous fungi. In two specific cases, where the internal intron interrupts the donor of the external intron after the first or after the second nucleotide, respectively, we show that intermediates with the sequence predicted by the "stwintron" hypothesis, are produced in the splicing process. This implies that two successive rounds of RNA scanning by the spliceosome are necessary to produce the mature mRNA. The phylogenetic distributions of the stwintrons we have identified suggest that they derive from "late" events, subsequent to the appearance of the host intron. They may well not be limited to fungal nuclear transcripts, and their generation and eventual disappearance in the evolutionary process are relevant to hypotheses of intron origin and alternative splicing.
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http://dx.doi.org/10.1016/j.fgb.2013.06.003DOI Listing
August 2013

Eisosome distribution and localization in the meiotic progeny of Aspergillus nidulans.

Fungal Genet Biol 2013 Apr 6;53:84-96. Epub 2013 Feb 6.

Institute of Biosciences and Applications, Microbial Molecular Genetics Laboratory, National Center for Scientific Research, Demokritos (NCSRD), Athens, Greece.

In the model filamentous fungus Aspergillus nidulans, PilA and PilB, two homologues of the Saccharomyces cerevisiae eisosome proteins Pil1/Lsp1, and SurG, a strict orthologue of Sur7, are assembled and form tightly packed structures in conidiospores. As A. nidulans differs in its reproduction pattern from the Saccharomycotina in that it has the ability to reproduce through two different types of spores, conidiospores and ascospores, the products of the asexual and the sexual cycle respectively, we investigated the eisosome distribution and localization during the sexual cycle. Our results show that core eisosome proteins PilA, PilB and SurG are not expressed in hülle cells or early ascospores, but are expressed in mature ascospores. All eisosomal proteins form punctate structures at the membrane of late ascospores. In mature but quiescent ascospores, PilA forms static punctate structures at the plasma membrane. PilB also was observed at the ascospore membrane as well, with higher concentration at the areas where the two halves of ascospores are joined together. Finally, SurG was localized both at the membrane of ascospores and perinuclearly. In germlings originating from ascospores the punctate structures were shown to be composed only of PilA. PilB is diffused in the cytoplasm and SurG was located in vacuoles and endosomes. This altered localization is identical to that found in germlings originated from conidiospores. In germinated ascospores PilA foci did not colocalise with the highly mobile and transient peripheral punctate structures of AbpA, a marker for sites of clathrin-mediated endocytosis. Deletions of each one or all the three core eisosomal genes do not affect viability or germination of ascospores. In the presence of myriocin - a specific inhibitor of sphingolipid biosynthesis - PilA-GFP foci of ascospore germlings were less numerous and their distribution was significantly altered, suggesting a correlation between PilA foci and sphingolipid biosynthesis.
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http://dx.doi.org/10.1016/j.fgb.2013.01.002DOI Listing
April 2013

FlbD, a Myb transcription factor of Aspergillus nidulans, is uniquely involved in both asexual and sexual differentiation.

Eukaryot Cell 2012 Sep 13;11(9):1132-42. Epub 2012 Jul 13.

Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.

In the fungus Aspergillus nidulans, inactivation of the flbA to -E, fluG, fluF, and tmpA genes results in similar phenotypes, characterized by a delay in conidiophore and asexual spore production. flbB to -D encode transcription factors needed for proper expression of the brlA gene, which is essential for asexual development. However, recent evidence indicates that FlbB and FlbE also have nontranscriptional functions. Here we show that fluF1 is an allele of flbD which results in an R47P substitution. Amino acids C46 and R47 are highly conserved in FlbD and many other Myb proteins, and C46 has been proposed to mediate redox regulation. Comparison of ΔflbD and flbD(R47P) mutants uncovered a new and specific role for flbD during sexual development. While flbD(R47P) mutants retain partial function during conidiation, both ΔflbD and flbD(R47P) mutants are unable to develop the peridium, a specialized external tissue that differentiates during fruiting body formation and ends up surrounding the sexual spores. This function, unique among other fluffy genes, does not affect the viability of the naked ascospores produced by mutant strains. Notably, ascospore development in these mutants is still dependent on the NADPH oxidase NoxA. We generated R47K, C46D, C46S, and C46A mutant alleles and evaluated their effects on asexual and sexual development. Conidiation defects were most severe in ΔflbD mutants and stronger in R47P, C46D, and C46S strains than in R47K strains. In contrast, mutants carrying the flbD(C46A) allele exhibited conidiation defects in liquid culture only under nitrogen starvation conditions. The R47K, R47P, C46D, and C46S mutants failed to develop any peridial tissue, while the flbD(C46A) strain showed normal peridium development and increased cleistothecium formation. Our results show that FlbD regulates both asexual and sexual differentiation, suggesting that both processes require FlbD DNA binding activity and that FlbD is involved in the response to nitrogen starvation.
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http://dx.doi.org/10.1128/EC.00101-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445977PMC
September 2012

Mutations in the basic loop of the Zn binuclear cluster of the UaY transcriptional activator suppress mutations in the dimerisation domain.

Fungal Genet Biol 2012 Sep 1;49(9):731-43. Epub 2012 Jul 1.

Microbiología, Instituto de Química Biológica, Universidad de la República, Casilla de Correo 1157, Montevideo, Uruguay.

UaY is the specific ZnII(2)Cys(6) transcriptional activator of the purine utilisation pathway in Aspergillus nidulans. Previous work has determined the consensus binding sequence by EMSA and foot-printing. We determine here that it binds as a dimer to its cognate CGG-N(6)-CCG sites. We identify the uaY109 mutation, which has been shown to affect differentially the expression of a number of UaY-regulated genes, as a F112I substitution in the DNA-binding motif dimerisation domain. We isolated back mutants, revertants carrying different residues at the same position (I112N and I112M) and intragenic suppressors mapping in the first loop of the Zn cluster (N75T and N75K). We have analysed the original mutant and its revertants by growth tests and by their effects on the mRNA steady states of five UaY-regulated genes. We have determined the effect of the different mutations on UaY dimerisation, on the apparent Kdiss of the UaY DNA-binding domain to appropriate DNA sequences and on the methylation interference pattern. We have attempted to rationalise these phenotypes by modelling the UaY DNA binding domain on the structure of the highly similar Ppr1p. However, modelling of the wild-type and mutant proteins provides only a partial explanation for the observed phenotypes. This suggests that the mutated residues may have other roles besides the obvious ones inferred from their position in the sequence and by the similarity of UaY and Ppr1p.
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http://dx.doi.org/10.1016/j.fgb.2012.06.009DOI Listing
September 2012

Aspergillus nidulans CkiA is an essential casein kinase I required for delivery of amino acid transporters to the plasma membrane.

Mol Microbiol 2012 May 11;84(3):530-49. Epub 2012 Apr 11.

Institut de Génétique et Microbiologie, Université Paris-Sud (XI), UMR 8621 CNRS 91450 Orsay, France.

Type I casein kinases are highly conserved among Eukaryotes. Of the two Aspergillus nidulans casein kinases I, CkiA is related to the δ/ε mammalian kinases and to Saccharomyces cerevisiae Hrr25p. CkiA is essential. Three recessive ckiA mutations leading to single residue substitutions, and downregulation using a repressible promoter, result in partial loss-of-function, which leads to a pleiotropic defect in amino acid utilization and resistance to toxic amino acid analogues. These phenotypes correlate with miss-routing of the YAT plasma membrane transporters AgtA (glutamate) and PrnB (proline) to the vacuole under conditions that, in the wild type, result in their delivery to the plasma membrane. Miss-routing to the vacuole and subsequent transporter degradation results in a major deficiency in the uptake of the corresponding amino acids that underlies the inability of the mutant strains to catabolize them. Our findings may have important implications for understanding how CkiA, Hrr25p and other fungal orthologues regulate the directionality of transport at the ER-Golgi interface.
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http://dx.doi.org/10.1111/j.1365-2958.2012.08042.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491690PMC
May 2012

The GATA factors AREA and AREB together with the co-repressor NMRA, negatively regulate arginine catabolism in Aspergillus nidulans in response to nitrogen and carbon source.

Fungal Genet Biol 2012 Mar 28;49(3):189-98. Epub 2012 Jan 28.

Institute of Genetics and Biotechnology, University of Warsaw, ul. Pawińskiego 5A, 02-106 Warsaw, Poland.

The filamentous fungus Aspergillus nidulans can utilize arginine both as a nitrogen and carbon source. Analysis of areA and areB single and double mutants has shown that the two GATA transcription factors AREA and AREB negatively regulate the expression of arginine catabolism genes agaA and otaA under nitrogen repressing conditions. AREA is necessary for the ammonium repression of agaA and otaA under carbon repressing conditions, while AREB is involved under carbon-limiting conditions. The ability of both AREA and AREB to sense the status of carbon metabolism is most probably dependent on NMRA, and not on the transcription factor CREA, which mediates general carbon catabolite repression in A. nidulans. NMRA is a co-repressor which has previously been shown to bind the C-terminus of AREA and inhibits its activity under conditions of nitrogen sufficiency, in response to high intracellular glutamine levels. We therefore propose a novel function for NMRA, the modulation of AREA and AREB activity in response to the carbon status of the cell.
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http://dx.doi.org/10.1016/j.fgb.2012.01.004DOI Listing
March 2012

The contribution of John Pateman to fungal genetics: a personal reminiscence.

Fungal Genet Biol 2011 Nov 4;48(11):1001-3. Epub 2011 Aug 4.

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http://dx.doi.org/10.1016/j.fgb.2011.07.010DOI Listing
November 2011