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Functions of protein SUMOylation remain incompletely understood in different cell types. Via forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO activation enzyme UbaB in the filamentous fungus Aspergillus nidulans. The ubaBQ247*, ΔubaB, and ΔsumO mutants all produce abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. The bridges are enclosed by nuclear membrane containing peripheral nuclear pore complex proteins that normally get dispersed during mitosis, and the bridges are also surrounded by cytoplasmic microtubules typical of interphase cells. Time-lapse sequences further indicate that most bridges persist through interphase prior to the next mitosis, and anaphase chromosome segregation can produce new bridges that persist into the next interphase. When the first mitosis happens at a higher temperature of 42°C, SUMOylation deficiency produces not only chromatin bridges but also many abnormally shaped single nuclei that fail to divide. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO targets being nuclear proteins. Finally, although the budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, loss of SUMOylation does not cause any obvious defect in dynein-mediated transport of nuclei and early endosomes, indicating that SUMOylation is unnecessary for dynein activation in A. nidulans. [ABSTRACT FROM AUTHOR]

An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences, all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best-studied elevator transporters is the UapA uric acid-xanthine/Hþ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead not only to increased rate of transport but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding and broadened specificity, leading in specific cases to UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function and how this knowledge can be applied to genetically modify their transport characteristics. [ABSTRACT FROM AUTHOR]

We report a novel sexual-cycle-specific gene-silencing system in the genetic model Aspergillus nidulans. Duplication of the mating type matA(HMG) gene in this haploid organism triggers Mat-induced silencing (MatIS) of both endogenous and transgenic matA genes, eliminates function of the encoded SRY structural ortholog, and results in formation of barren fruiting bodies. MatIS is spatiotemporally restricted to the prezygotic stage of the sexual cycle and does not interfere with vegetative growth, asexual reproduction, differentiation of early sexual tissues, or fruiting body development. MatIS is reversible upon deletion of the matA transgene. In contrast to other sex-specific silencing phenomena, MatIS silencing has nearly 100% efficiency and appears to be independent of homologous duplicated DNA segments. Remarkably, transgene-derived matA RNA might be sufficient to induce MatIS. A unique feature of MatIS is that RNA-mediated silencing is RNA interference/Argonaute-independent and is restricted to the nucleus having the duplicated gene. The silencing phenomenon is recessive and does not spread between nuclei within the common cytoplasm of a multinucleate heterokaryon. Gene silencing induced by matA gene duplication emerges as a specific feature associated with matA(HMG) regulation during sexual development.

Cytokinesis, as the final step of cell division, plays an important role in fungal growth and proliferation. In the filamentous fungus Aspergillus nidulans, defective cytokinesis is able to induce abnormal multinuclear or nonnucleated cells and then result in reduced hyphal growth and abolished sporulation. Previous studies have reported that a conserved contractile actin ring (CAR) protein complex and the septation initiation network (SIN) signaling kinase cascade are required for cytokinesis and septation; however, little is known about the role(s) of scaffold proteins involved in these two important cellular processes. In this study, we show that a septumlocalized scaffold protein paxillin B (PaxB) is essential for cytokinesis/septation in A. nidulans. The septation defects observed in a paxB deletion strain resemble those caused by the absence of another identified scaffold protein, a-actinin (AcnA). Deletion of a-actinin (AcnA) leads to undetectable PaxB at the septation site, whereas deletion of paxB does not affect the localization of a-actinin at septa. However, deletion of either a-actinin (acnA) or paxB causes the actin ring to disappear at septation sites during cytokinesis. Notably, overexpression of a-actinin acnA partially rescues the septum defects of the paxB mutant but not vice versa, suggesting AcnA may play a dominant role over that of PaxB for cytokinesis and septation. In addition, PaxB and a-actinin affect the septal dynamic localization of MobA, a conserved component of the SIN pathway, suggesting they may affect the SIN protein complex function at septa. Protein pulldown assays combined with liquid chromatography-mass spectrometry identification indicate that a-actinin AcnA and PaxB likely do not directly interact, but presumably belong to an actin cytoskeleton protein network that is required for the assembly and contraction of the CAR. Taken together, findings in this study provide novel insights into the roles of conserved scaffold proteins during fungal septation in A. nidulans. [ABSTRACT FROM AUTHOR]

Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system. [ABSTRACT FROM AUTHOR]

Functions of protein SUMOylation remain incompletely understood in different cell types. Via forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO activation enzyme UbaB in the filamentous fungus Aspergillus nidulans. The ubaBQ247*, ΔubaB, and ΔsumO mutants all produce abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. The bridges are enclosed by nuclear membrane containing peripheral nuclear pore complex proteins that normally get dispersed during mitosis, and the bridges are also surrounded by cytoplasmic microtubules typical of interphase cells. Time-lapse sequences further indicate that most bridges persist through interphase prior to the next mitosis, and anaphase chromosome segregation can produce new bridges that persist into the next interphase. When the first mitosis happens at a higher temperature of 42°C, SUMOylation deficiency produces not only chromatin bridges but also many abnormally shaped single nuclei that fail to divide. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO targets being nuclear proteins. Finally, although the budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, loss of SUMOylation does not cause any obvious defect in dynein-mediated transport of nuclei and early endosomes, indicating that SUMOylation is unnecessary for dynein activation in A. nidulans., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Published by Oxford University Press on behalf of The Genetics Society of America 2023.)

Neurospora crassa is an established reference organism to investigate carotene biosynthesis and light regulation. However, there is little evidence of its capacity to produce secondary metabolites. Here, we report the role of the fungal-specific regulatory velvet complexes in development and secondary metabolism (SM) in N. crassa. Three velvet proteins VE-1, VE-2, VOS-1, and a putative methyltransferase LAE-1 show light-independent nucleocytoplasmic localization. Two distinct velvet complexes, a heterotrimeric VE-1/VE-2/LAE-1 and a heterodimeric VE-2/VOS-1 are found in vivo. The heterotrimer-complex, which positively regulates sexual development and represses asexual sporulation, suppresses siderophore coprogen production under iron starvation conditions. The VE-1/VE-2 heterodimer controls carotene production. VE-1 regulates the expression of .15% of the whole genome, comprising mainly regulatory and developmental features. We also studied intergenera functions of the velvet complex through complementation of Aspergillus nidulans veA, velB, laeA, vosA mutants with their N. crassa orthologs ve-1, ve-2, lae-1, and vos-1, respectively. Expression of VE-1 and VE-2 in A. nidulans successfully substitutes the developmental and SM functions of VeA and VelB by forming two functional chimeric velvet complexes in vivo, VelB/VE-1/LaeA and VE-2/VeA/LaeA, respectively. Reciprocally, expression of veA restores the phenotypes of the N. crassa ve-1 mutant. All N. crassa velvet proteins heterologously expressed in A. nidulans are localized to the nuclear fraction independent of light. These data highlight the conservation of the complex formation in N. crassa and A. nidulans. However, they also underline the intergenera similarities and differences of velvet roles according to different life styles, niches and ontogenetic processes. [ABSTRACT FROM AUTHOR]

FurE, a member of the Nucleobase Cation Symporter 1 transporter family in Aspergillus nidulans, is specific for allantoin, uric acid (UA), uracil, and related analogs. Herein, we show that C- or N-terminally-truncated FurE transporters (FurE-ΔC or FurE-Δ) present increased protein stability, but also an inability for UA transport. To better understand the role of cytoplasmic terminal regions, we characterized genetic suppressors that restore FurE-ΔC-mediated UA transport. Suppressors map in the periphery of the substratebinding site [Thr133 in transmembrane segment (TMS)3 and Val343 in TMS8], an outward-facing gate (Ser296 in TMS7, Ile371 in TMS9, and Tyr392 and Leu394 in TMS10), or in flexible loops (Asp26 in LN, Gly222 in L5, and Asn308 in L7). Selected suppressors were also shown to restore the wild-type specificity of FurE-ΔN, suggesting that both C- and/or N-terminal domains are involved in intramolecular dynamics critical for substrate selection. A direct, substrate-sensitive interaction of C- and/or N-terminal domains was supported by bimolecular fluorescence complementation assays. To our knowledge, this is the first case where not only the function, but also the specificity, of a eukaryotic transporter is regulated by its terminal cytoplasmic regions. [ABSTRACT FROM AUTHOR]

Ontogenetic phases separating growth from reproduction are a common feature of cellular life. Long recognized for flowering plants and animals, early literature suggests this life-history component may also be prevalent among multicellular fungi. We establish the basis of developmental competence--the capacity to respond to induction of asexual development--in the filamentous saprotroph Aspergillus nidulans, describing environmental influences, including genotype-by-environment interactions among precocious mutants, gene expression associated with wild type and precocious competence acquisition, and the genetics of competence timing. Environmental effects are consistent with a threshold driven by metabolic rate and organism density, with pH playing a particularly strong role in determining competence timing. Gene expression diverges significantly over the competence window, despite a lack of overt morphological change, with differentiation in key metabolic, signaling, and cell trafficking processes. We identify five genes for which mutant alleles advance competence timing, including the conserved GTPase RasB (AN5832) and ambient pH sensor PalH (AN6886). In all cases examined, inheritance of competence timing is complex and non-Mendelian, with F1 progeny showing highly variable transgressive timing and dominant parental effects with a weak contribution from progeny genotype. Competence provides a new model for nutrient-limited life-cycle phases, and their elaboration from unicellular origins. Further work is required to establish the hormonal and bioenergetic basis of the trait across fungi, and underlying mechanisms of variable inheritance. [ABSTRACT FROM AUTHOR]

Carbon catabolite repression (CCR) is a process that selects the energetically most favorable carbon source in an environment. CCR represses the use of less favorable carbon sources when a better source is available. Glucose is the preferential carbon source for most microorganisms because it is rapidly metabolized, generating quick energy for growth. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, a C2H2 finger domain DNA-binding protein. The aim of this work was to investigate the regulation of CreA and characterize its functionally distinct protein domains. CreA depends in part on de novo protein synthesis and is regulated in part by ubiquitination. CreC, the scaffold protein in the CreB-CreC deubiquitination (DUB) complex, is essential for CreA function and stability. Deletion of select protein domains in CreA resulted in persistent nuclear localization and target gene repression. A region in CreA conserved between Aspergillus spp. and Trichoderma reesei was identified as essential for growth on various carbon, nitrogen, and lipid sources. In addition, a role of CreA in amino acid transport and nitrogen assimilation was observed. Taken together, these results indicate previously unidentified functions of this important transcription factor. These novel functions serve as a basis for additional research in fungal carbon metabolism with the potential aim to improve fungal industrial applications. [ABSTRACT FROM AUTHOR]

In the model fungus Aspergillus nidulans, asexual development is induced from vegetative hyphae by a set of early regulators including the bZIP-type transcription factor FlbB. To determine the range of genes under the influence of the transcriptional activity of FlbB and to characterize their role in fungal development, we sequenced and compared the transcriptomes of a DflbB mutant and its isogenic wild-type strain at different developmental stages. Results confirmed the activating role of FlbB on downstream regulators of conidiation such as flbD and brlA. However, FlbB has additional functions beyond the induction of asexual development. Among the changes observed, absence of a functional FlbB caused induction of the dba cluster and synthesis of a secondary metabolite with bactericidal properties. In addition, a new transcriptional target of FlbB was unveiled, urdA, that codes for a putative transcription factor that represses premature sexual development. Taken together, our results indicate that the activators of asexual development simultaneously exert a role on other cellular functions, including an inhibitory effect on the sexual cycle, and reinforce the hypothesis that mutually exclusive metabolic and cellular patterns are associated with different morphogenetic programs. [ABSTRACT FROM AUTHOR]

Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein-protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2cdc2 that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2cdc2, nimE6cyclinB, and nimT23cdc25, but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10cyclinB mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxAHrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and DsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXAHRB1 is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2CDC2/NIMECYCLINB kinase activity and that snxA1 or ΔsnxA alter localization patterns of NIMECYCLINB at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway. [ABSTRACT FROM AUTHOR]

Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast. [ABSTRACT FROM AUTHOR]

Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (-) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (-) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening > 100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA~flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the DfluG, DflbA, and DflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA. [ABSTRACT FROM AUTHOR]

The nuclear pore complex proteins SonA and SonB, the orthologs of mammalian RAE1 and NUP98, respectively, were identified in Aspergillus nidulans as cold-sensitive suppressors of a temperature-sensitive allele of the essential mitotic NIMA kinase (nimA1). Subsequent analyses found that sonB1 mutants exhibit temperature-dependent DNA damage sensitivity. To understand this pathway further, we performed a genetic screen to isolate additional conditional DNA damage-sensitive suppressors of nimA1. We identified two new alleles of SonA and four intragenic nimA mutations that suppress the temperature sensitivity of the nimA1 mutant. In addition, we identi ed SonC, a previously unstudied binuclear zinc cluster protein involved with NIMA and the DNA damage response. Like sonA and sonB, sonC is an essential gene. SonC localizes to nuclei and partially disperses during mitosis. When the nucleolar organizer region (NOR) undergoes mitotic condensation and removal from the nucleolus, nuclear SonC and histone H1 localize in a mutually exclusive manner with H1 being removed from the NOR region and SonC being absent from the end of the chromosome beyond the NOR. This region of chromatin is adjacent to a cluster of nuclear pore complexes to which NIMA localizes last during its progression around the nuclear envelope during initiation of mitosis. The results genetically extend the NIMA regulatory system to include a protein with selective large-scale chromatin location observed during mitosis. The data suggest a model in which NIMA and SonC, its new chromatin-associated suppressor, might help to orchestrate global chromatin states during mitosis and the DNA damage response. [ABSTRACT FROM AUTHOR]

Sexual reproduction is a fundamental developmental process that allows for genetic diversity through the control of zygote formation, recombination, and gametogenesis. The correct regulation of these events is paramount. Sexual reproduction in filamentous fungi, including mating strategy (self-fertilization/homothallism or outcrossing/heterothallism), is determined by the expression of mating type genes at mat loci. Aspergillus nidulans matA encodes a critical regulator that is a fungal ortholog of the hSRY/SOX9 HMG box proteins. In contrast to well-studied outcrossing systems, the molecular basis of homothallism and role of mating type genes during a self-fertile sexual cycle remain largely unknown. In this study the genetic model organism, A. nidulans, has been used to investigate the regulation and molecular functions of the matA mating type gene in a homothallic system. Our data demonstrate that complex regulatory mechanisms underlie functional matA expression during self-fertilization and sexual reproduction in A. nidulans. matA expression is suppressed in vegetative hyphae and is progressively derepressed during the sexual cycle. Elevated levels of matA transcript are required for differentiation of fruiting bodies, karyogamy, meiosis, and efficient formation of meiotic progeny. matA expression is driven from both initiator (Inr) and novel promoter elements that are tightly developmentally regulated by position-dependent and position-independent mechanisms. Deletion of an upstream silencing element, matA SE, results in derepressed expression from wild-type (wt) promoter elements and activation of an additional promoter. These studies provide novel insights into the molecular basis of homothallism in fungi and genetic regulation of sexual reproduction in eukaryotes. [ABSTRACT FROM AUTHOR]

Light regulates several aspects of the biology of many organisms, including the balance between asexual and sexual development in some fungi. To understand how light regulates fungal development at the molecular level we have used Aspergillus nidulans as a model. We have performed a genome-wide expression analysis that has allowed us to identify >400 genes upregulated and >100 genes downregulated by light in developmentally competent mycelium. Among the upregulated genes were genes required for the regulation of asexual development, one of the major biological responses to light in A. nidulans, which is a pathway controlled by the master regulatory gene brlA. The expression of brlA, like conidiation, is induced by light. A detailed analysis of brlA light regulation revealed increased expression after short exposures with a maximum after 60 min of light followed by photoadaptation with longer light exposures. In addition to brlA, genes flbA-C and fluG are also light regulated, and flbA-C are required for the correct light-dependent regulation of the upstream regulator fluG. We have found that light induction of brlA required the photoreceptor complex composed of a phytochrome FphA, and the white-collar homologs LreA and LreB, and the fluffy genes flbA-C. We propose that the activation of regulatory genes by light is the key event in the activation of asexual development by light in A. nidulans. [ABSTRACT FROM AUTHOR]

The ability of fungi to generate polarized cells with a variety of shapes likely reflects precise temporal and spatial control over the formation of polarity axes. The bud site selection system of Saccharomyces cerevisiae represents the best-understood example of such a morphogenetic regulatory system. However, the extent to which this system is conserved in the highly polarized filamentous fungi remains unknown. Here, we describe the functional characterization and localization of the Aspergillus nidulans homolog of the axial bud site marker Bud3. Our results show that AnBud3 is not required for polarized hyphal growth per se, but is involved in septum formation. In particular, our genetic and biochemical evidence implicates AnBud3 as a guanine nucleotide exchange factor for the GTPase Rho4. Additional results suggest that the AnBud3-Rho4 module acts downstream of the septation initiation network to mediate recruitment of the formin SepA to the site of contractile actin ring assembly. Our observations provide new insight into the signaling pathways that regulate septum formation in filamentous fungi. [ABSTRACT FROM AUTHOR]

XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, xprG2 revertants were isolated and shown to carry mutations in genes which we have named sogA-C (suppressors of xprG). The translocation breakpoint in the sogA1 mutant was localized to a homolog of Saccharomyces cerevisiae VPS5 and mapping data indicated that sogB was tightly linked to a VPS17homolog. Complementation of the sogAl and sogB1 mutations and identification of nonsense mutations in the sogA2and sogB1 alleles confirmed the identification. Vpsi 7p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in sogA-C permit secretion of intracellular proteases, as in S. cerevisiae vps5 and vps17mutants. In contrast to S. cerevisiae, the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG- independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants. [ABSTRACT FROM AUTHOR]

The uaY gene encodes the transcriptional activator of purine catabolism genes in Aspergillus nidulans. uaY12 results in strongly defective growth on purines as nitrogen sources and in strongly diminished transcription of UaY-regulated genes. This mutation introduces an ATG codon 64 hp upstream of the uaY ATG, generating a 68-codon open reading frame (uORFA), overlapping with the uaY ORF. uaY12 revertants fall into three categories: i. The majority eliminate the aberrant ATG. The growth and transcriptional phenotypes of these revertants are identical to those of the wild type. ii. Two revertants create a stop codon in frame with the uaY12 aberrant ATG, shortening the length of the uORFA, thus uORFA no longer overlaps the uaY ORF. The latter are partial suppressors of the uaY12 mutation, while chain termination suppressors, in turn, suppress this novel phenotype. iii. Two partial suppressors are unlinked to uaY These two mutations result in a pleiotropic phenotype usually associated with ribosomal proteins. We hypothesize that uORFA strongly diminishes translation of the uaYORF and that 1-evertants negate this effect by a number of different mechanisms. The first-AUG rule and the phenomena of translational inhibition and leaky scanning provide a coherent explanation of the results presented in this article. [ABSTRACT FROM AUTHOR]

In fungal hyphae, apical dominance refers to the suppression of secondary polarity axes in the general vicinity of a growing hyphal tip. The mechanisms underlying apical dominance remain largely undefined, although calcium signaling may play a role. Here, we describe the localized accumulation of reactive oxygen species (ROS) in the apical region of Aspergillus niclulans hyphae. Our analysis of atmA (ATM) and prpA (PARP) mutants reveals a correlation between localized production of ROS and enforcement of apical dominance. We also provide evidence that NADPH oxidase (Nox) or related flavoproteins are responsible for the generation of ROS at hyphal tips and characterize the roles of the potential Nox regulators NoxR, RacL and Cdc42 in this process. Notably, our genetic analyses suggest that Raci activates Nox, whereas NoxR and Cdc42 may function together in a parallel pathway that regulates Nox localization. Moreover, the latter pathway may also include Bemi, which we propose represents a p40phox analog in fungi. Collectively, our results support a model whereby localized Nox activity generates a pool of ROS that defines a dominant polarity axis at hyphal tips. [ABSTRACT FROM AUTHOR]

Peroxisomes are organdies containing a diverse array of enzymes. In fungi they are important for carbon source utilization, pathogenesis, development, and secondary metabolism. We have studied Aspergillus nidulans peroxin (pex) mutants isolated by virtue of their inability to grow on hutyrate or by the inactivation of specific pex genes. While all pex mutants are able to form colonies, those unable to import PTSI proteins are partially defective in asexual and sexual development. The pex mutants are able to grow on acetate but are affected in growth on fatty acids, indicating a requirement for the peroxisomal localization of β-oxidation enzymes. However, mislocalization of malate synthase does not prevent growth on either fatty acids or acetate, showing that the glyoxylate cycle does not require peroxisomal localization. Proliferation of peroxisomes is dependent on fatty acids, hut not on acetate, and on PexK (Pex11), expression of which is activated by the FarA transcription factoc Proliferation was greatly i-educed in afarAΔ strain. A mutation affecting a mitochoclrial ketoacyl-CoA thiolase and disruption of a mitochondrial hydroxy-acyl-CoA dehydrogenase gene prevented growth on short-chain but not long-chain fatty acids. Together with previous results, this is consistent with growth on even-numbered short-chain fatty acids requiring a mitochondrial as well as a peroxisomal β-oxidation pathway. The mitochondrial pathway is not required for growth on valerate or for long-chain fatty acid utilization. [ABSTRACT FROM AUTHOR]

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmAATM loss of function caused synthetic lethality when combined with mutation in UvsBATR. Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkACHK1 and chkBCHK2 genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for ΔatmA, ΔuvsB, ΔchkA, and ΔchkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AlmA, and bimEAPC1, ankAWEE1 and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents. [ABSTRACT FROM AUTHOR]

The author reflects on the concepts of Guido Pontecorvo on genetics. He discusses the contributions of Pontecorvo on genetics, while being a refugee from fascism. Some of his discussions on Pontecorvo's contributions include his doctoral project on the sterility of melanogaster and simulan hybrids, as well as on his pioneering work in microbial genetics which found Aspergillus nidulans as genetically tractable model organism.

Sphingolipid C4 hydroxylase catalyzes the conversion of dihydrosphingosine to phytosphingosine. In Saccharmnyces cerevisiae, Sur2 is essential for sphingolipid C4 hydroxylation activity but not essential for normal growth. Here we demonstrate that the Aspergillus nidulans Sur2 homolog BasA is also required for phytosphingosine biosynthesis but is also essential for viability. We previously reported that a point missense mutation in basA resulted in aberrant cell wall thickening. Here our data suggest that accumulation of dihydrosphingosine is responsible for this phenotype. In addition, two different mutations in basA consistently accelerated the transition from asexual development to sexual development compared to the wild-type strain. The phenotype could be suppressed by exogenous addition of phytosphingosine. Northern analysis suggests that faster sexual development in the basA mutant might be due to a higher transcription leve. of ppoA and steA, genes demonstrated to coordinate a balance between asexual and sexual development in A. nidulans. Consistent with these findings, mutations in the ceramide-synthase-encoding genes larA and lagA also caused faster transition from asexual to sexual development, supporting the involvement of sphingolipid metabolism in fungal morphogenesis. [ABSTRACT FROM AUTHOR]

Aspergillus nidulans can utilize carbon sources that result in the production of TCA cycle intermediates, thereby requiring gluconeogenesis. We have cloned the acuG gene encoding fructose-1,6 bisphosphatase and fount! that expression of this gene is regulated by carbon catabolite repression as well as by induction by a TCA cycle intermediate similar to the induction of the previously studied acuF gene encoding phosphoenolpyruvate carboxykinase. The acuN356 mutation results in loss of growth on gluconeogenic carbon sources. Cloning of acuN has shown that it encodes enolase, an enzyme involved in both glycolysis and gluconeogenesis. The acuN356 mutation is a translocation with a breakpoint in the 5′ untranslated region resulting in loss of expression in response to gluconeogenic but not glycolytic carbon sources. Mutations in the acuK and acuM genes affect growth on carbon sources requiring gluconeogenesis and result in loss of induction of the acuF, acuN, and acuG genes by sources of TCA cycle intermediates. Isolation anti sequencing of these genes has shown that they encode proteins with similar but distinct Zn(2) Cys(6) DNA-binding domains, suggesting a direct role in transcriptional control of gluconeogenic genes. These genes are conserved in other filamentous ascomycetes, indicating their significance for the regulation of carbon source utilization. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) is embedded in the nuclear envelope where it mediates transport between the cytoplasm and nucleus and helps to organize nuclear architecture. We previously isolated sonB1, a mutation encoding a single amino acid substitution within the Aspergillus nidulans SONBnNup98 NPC protein (nucleoporin). Here we demonstrate that this mutation causes marked DNA damage sensitivity at 42°. Although SONBnNup98 has roles in the G2 transition, we demonstrate that the G2 DNA damage checkpoint is functional in the sonB1 mutant at 42°. The MRN complex is composed of MRE11, RAD50, and NBS1 and functions in checkpoint signaling, DNA repair, and telomere maintenance. At 42° we find that the DNA damage response defect of sonB1 mutants causes synthetic lethality when combined with mutations in scaANBS1, the A. nidulans homolog of NBS1. We provide evidence that this synthetic lethality is independent of MRN cell cycle checkpoint functions or MREAMRE11-rnediated DNA repair functions. We also demonstrate that the single A. nidulans histone H2A gene contains the C-terminal SQE motif of histone H2AX isoforms and that this motif is required for the DNA damage response. We propose that the sonB1 nucleoporin mutation causes a defect in a novel part of the DNA damage response. [ABSTRACT FROM AUTHOR]

The kinase cascade of the septation initiation network (SIN), first revealed in fission yeast, activates the contraction of the actomyosin ring, and plays an essential role in fungal septation. Mob1p, an evolutionarily conserved SIN protein, is associated with the most downstream kinase of this cascade in fission yeast. In this study, the mobA gene encoding a homologous protein was isolated from the filamentous fungus Aspergillus nidulans, whose mycelium is made of multinucleate cells. The MOBA protein was required for septation and conidiation, hut was not essential for hyphal extension and colony formation. To identify genes that act antagonistically against the SIN, UV mutagenesis was carried out to isolate suppressor (smo) mutations that restored conidiation when MOBA was not expressed. Microscopic examination indicated that the restored conidiation was concomitant with restored septation in the absence of the MOBA protein. Eight recessive smo mutations in five complementation groups also bypassed the requirement of the SIN kinases SEPH and SIDB for septum formation and conidiation. However, none of these smo mutations affected the localization of MOBA. Among smo mutations, smoA and smoB mutations caused reduced hyphal growth and colony formation. They also rendered hypersensitivity to low doses of the microtubule-depolymerizing agent benomyl for conidiation. Therefore, in A. nidulans, proteins encoded by the smo genes likely have an antagonistic interaction against the SIN pathway to regulate septation and conidiation. [ABSTRACT FROM AUTHOR]

Ataxia telangiectasia (A-T) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. The most prominent clinical feature observed in A-T patients is the de- generation of Purkinje motor neurons. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. However, in Purkinje cells, the bulk of ATM localizes to the cytoplasm and may play a role in vesicle trafficking. The nature of this function, and its involvement in the pathology underlyingA-T, remain unknown. Here we characterize the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we find that AtmA is also required for polarized hyphal growth. We demonstrate that an atmA mutant fails to generate a stable axis of hyphal polarity. Notably, cytoplasmic microtubules display aberrant cortical interactions at the hyphal tip. Our results suggest that AtmA regulates the function and/or localization of landmark proteins required for the formation of a polarity axis. We propose that a similar function may contribute to the establishment of neuronal polarity. [ABSTRACT FROM AUTHOR]

The asexual spore is one of the most crucial factors contributing to the fecundity and fitness of filamentous fungi. Although the developmental activator FluG was shown to he necessary for activation of asexual sporulation (conidiation) and production of the carcinogenic mycotoxin sterigmatocystin (ST) in the model filamentous fungus Aspergillus nidulans, the molecular mechanisms underlying the developmental switch have remained elusive. In this study, we report that the FIuG-mediated conidiation in A. nidulans occurs via derepression. Suppressor analyses of fluG led to the identification of the sfgA gene encoding a novel protein with the Gal4-type Zn(II)2Cys6 binuclear cluster DNA-binding motif at the N terminus. Deletion (Δ) and 31 other loss-of-function sfgA mutations bypassed the need for fluG in conidiation and production of ST. Moreover, both ΔsfgA and ΔsfgA ΔfluG mutations resulted in identical phenotypes in growth, conidiation, and ST production, indicating that the primary role of FluG is to remove repressive effects imposed by SfgA. In accordance with the proposed regulatory role of SfgA, overexpression of sfgA inhibited conidiation and delayed/reduced expression of conidiation- and ST-specific genes. Genetic analyses demonstrated that SfgA functions downstream of FluG hut upstream of transcriptional activators (FIbD, FlbC, FIbB, and BrlA) necessary for normal conidiation. [ABSTRACT FROM AUTHOR]

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nku∧Δ) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nku∧Δ and the heterologous selectable markers make up a very efficient gene- targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible. [ABSTRACT FROM AUTHOR]

The signalosome (CSN) is a conserved multiprotein complex involved in regulation of eukaryotic development and is also required to activate ribonucleotide reductase for DNA synthesis. In Aspergillus nidulans, csnD/csnE are key regulators of sexual development. Here, we investigated whether the csnD/csnE genes are involved in the DNA damage response in this fungus. The growth of the csnD/csnE deletion mutants was reduced by subinhibitory concentrations of hydroxyurea, camptothecin, 4-nitroquinoline oxide, and methyl methanesulfonate. A. nidulans increases csnD/csnE mRNA levels when it is challenged by different DNA-damaging agents. There is no significant transcriptional induction of the csnE promoter fused with lacZgene in the presence of DNA-damaging agents, suggesting that increased mRNA accumulation is due to increased mRNA stability. Septation was not inhibited in the csnD/csnE deletion mutants while ΔuvsB ΔcsnE presented an increase in septation upon DNA damage caused by methyl methanesulfonate, suggesting that uvsBATR and csnE genetically interact during checkpoint-dependent inhibition of septum formation. The double ΔcsnD/ΔcsnE ΔnpkA mutants were more sensitive to DNA-damaging agents than were the respective single mutants. Our results suggest that csnD/csnE genes are involved in the DNA damage response and that NpkA and UvsBATR genetically interact with the signalosome. [ABSTRACT FROM AUTHOR]

A rapid route to gene molecular identification involves using recombination frequencies in locating mutational sequence changes. We describe a case where the recombination frequency is deceptively low, probably reflecting centromere proximity. Recombination frequencies are greatly reduced near the centromeres on the right arms of chromosomes Ill and IV of Aspergillus nidulans. [ABSTRACT FROM AUTHOR]

The alkaline ambient pH signal transduction pathway component PalC has no assigned molecular role. Therefore we attempted a gene-specific mutational analysis and obtained 55 new palC loss-of-function alleles including 94 single residue substitutions. Refined similarity searches reveal conserved PalC regions including one with convincing similarity to the BRO1 domain, denoted PCBROH, where clustering of mutational changes, including PCBROH key residue substitutions, supports its structural and/or functional importance. Since the BRO1 domain occurs in the multivesicular body (MVB) pathway protein Bro1/Vps31 and also the pH signal transduction protein PalA (Rim20), both of which interact with MVB component (ESCRT-III protein) Vps32/Snf7, this might reflect a further link between the pH response and endocytosis. [ABSTRACT FROM AUTHOR]

Vegetative growth signaling in the filamentous fungus Aspergillus nidulans is primarily mediated by the heterotrimeric G-protein composed of FadA (Gα), SfaD (Gβ), and a presumed Gγ. Analysis of the A. nidulans genome identified a single gene named gpgA encoding a putative Gγ-subunit. The predicted GpgA protein consists of 90 amino acids showing 72% similarity with yeast Ste18p. Deletion (Δ) of gpgA resulted in restricted vegetative growth and lowered asexual sporulation. Moreover, similar to the ΔsfaD mutant, the ΔgpgA mutant was unable to produce sexual fruiting bodies (cleistothecia) in self-fertilization and was severely impaired with cleistothecial development in outcross, indicating that both SfaD and GpgA are required for fruiting body formation. Developmental and morphological defects caused by deletion of flbA encoding an RGS protein negatively controlling FadA-mediated vegetative growth signaling were suppressed by ΔgpgA, indicating that GpgA functions in FadA-SfaD-mediated vegetative growth signaling. However, deletion of gpgA could not bypass the need for the early developmental activator FluG in asexual sporulation, suggesting that GpgA functions in a separate signaling pathway. We propose that GpgA is the only A. nidulans Gγ-subunit and is required for normal vegetative growth as well as proper asexual and sexual developmental progression. [ABSTRACT FROM AUTHOR]

The role of heterotrimeric G-proteins in cAMP-dependent germination of conidia was investigated in the filamentous ascomycete Aspergillus nidulans. We demonstrate that the Gα-subunit GanB mediates a rapid and transient activation of cAMP synthesis in response to glucose during the early period of germination. Moreover, deletion of individual G-protein subunits resulted in defective trehalose mobilization and altered germination kinetics, indicating that GanB(α)-SfaD(β)-GpgA(γ) constitutes a functional heterotrimer and controls cAMP/PKA signaling in response to glucose as well as conidial germination. Further genetic analyses suggest that GanB plays a primary role in cAMP/PKA signaling, whereas the SfaD-GpgA (Gβγ) heterodimer is crucial for proper activation of GanB signaling sensitized by glucose. In addition, the RGS protein RgsA is also involved in regulation of the cAMP/PKA pathway anti germination via attenuation of GanB signaling. Genetic epistatic analyses led us to conclude that all controls exerted by GanB(α)-SfaD(β)-GpgA(γ) on conidial germination are mediated through the cAMP/PKA pathway. Furthermore, GanB may function in sensing various carbon sources and subsequent activation of downstream signaling for germination. [ABSTRACT FROM AUTHOR]

Evidence in several microorganisms indicates that Amt proteins are gas channels for NH3 and CH3NH2, and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity. All 16 spontaneous methylammonium-resistant mutants that we analyzed had defects in accumulation of [14C] methylammonium. Genetic crosses indicated that 12 had lesions in a single locus, whereas two each had lesions in other loci. Lesions in different loci were correlated with different degrees of defect in [14C]methylammonium uptake. One mutant in the largest class had an insert in the AMT4 gene, and the insert cosegregated with methylammonium resistance in genetic crosses. The other 11 strains in this class also had amt4 lesions, which we characterized at the molecular level. Properties of the amt4 mutants were clearly different from those of rh1 RNAi lines. They indicated that the physiological substrates for Amt and Rh proteins, the only two members of their protein superfamily, are NH3 and CO3, respectively. [ABSTRACT FROM AUTHOR]

SepB is an essential, conserved protein required for chromosomal DNA metabolism in Aspergillus nidulans. Homologs of SepB include yeast Ctf4p and human hAnd-1. Molecular and bioinformatic characterization of these proteins suggests that they act as molecular scaffolds. Furthermore, recent observations implicate the yeast family members in lagging-strand replication and the establishment of sister-chromatid cohesion. Here, we demonstrate that SepB functions in the A. nidulans DNA damage response. In particular, analysis of double mutants reveals that SepB is a member of the UvsCRAD51 epistasis group. In accord with this prediction, we show that UvsCRAD51 forms DNA-damage-induced nuclear foci in a manner that requires SepB function. We also provide evidence that implicates SepB in sister-chromatid cohesion, thereby suggesting that cohesion may play a role in regulating tile localization and/or assembly of UvsCRAD51 complexes. [ABSTRACT FROM AUTHOR]

The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166–173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA. [ABSTRACT FROM AUTHOR]

The versatility of RNA-dependent RNA polymerases (RDRPs) in eukaryotic gene silencing is perhaps best illustrated in the kingdom Fungi. Biochemical and genetic studies of Schizosaccharomyces pombe and Neurospora crassa show that these types of enzymes are involved in a number of fundamental gene-silencing processes, including heterochromatin regulation and RNA silencing in S. pombe and meiotic silencing and RNA silencing in N. crassa. Here we show that Aspergillus nidulans, another model fungus, does not require an RDRP for inverted repeat transgene (IRT)-induced RNA silencing. However, RDRP requirements may vary within the Aspergillus genus as genomic analysis indicates that A. nidulans, but not A. fumigatus or A. oryzae, has lost a QDE-1 ortholog, an RDRP associated with RNA silencing in N. crassa. We also provide evidence suggesting that 5′ → 3′ transitive RNA silencing is not a significant aspect of A. nidulans IRT- RNA silencing. These results indicate a lack of conserved kingdom-wide requirements for RDRPs in fungal RNA silencing. [ABSTRACT FROM AUTHOR]

Here we have characterized the putative Zn(II)2Cys6 transcription factor Rosa from the filamentous fungus Aspergillus nidulans. The rosA gene encodes a protein of 713 aa, which shares 38% sequence similarity to Prol from Sordaria macrospora. In contrast to Prol, which promotes the transition from protoperithecia to perithecia, RosA is a negative regulator of sexual development in A. nidulans. Transcript levels of rosA were usually very low and were only transiently upregulated upon carbon starvation and at 12 hr of asexual development. Deletion of rosA only slightly induced fruiting-body formation under standard culture conditions, but enabled sexual development under low-glucose and high-osmolarity conditions and the production of Hülle cells under submersed growth conditions. Stimulation of fruiting-body formation on agar surfaces was dependent on veA. In ΔrosA strains, transcript levels of the sexual developmental regulators nsdD, veA, and stuA were increased. Overexpression of rosA led to a reduction of hyphal growth and to a fluffy phenotype. Post-transcriptional regulation of RosA, with a regulated accumulation in the nucleus, was shown using a RosA-GFP fusion protein. We propose that RosA represses sexual development upon integration of several environmental signals. [ABSTRACT FROM AUTHOR]

Propionyl-CoA is an intermediate metabolite produced through a variety of pathways including thioesterifIcation of propionate and catabolism of odd chain fatty acids and select amino acids. Previously, we found that disruption of the methylcitrate synthase gene, mcsA, which blocks propionyl-CoA utilization, as well as growth on propionate impaired production of several polyketides-molecules typically derived from acetyl-CoA and malonyl-CoA-including sterigmatocystin (ST), a potent carcinogen, and the conidiospore pigment. Here we describe three lines of evidence that demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis. First, inactivation of a putative propionyl-CoA synthase, PcsA, which converts propionate to propionyl-CoA, restored polyketide production and reduced cellular propionyl-CoA content in a ΔmcsA background. Second, inactivation of the acetyl-CoA synthase, FacA, which is also involved in propionate utilization, restored polyketide production in the Amos background. Third, fungal growth on several compounds (e.g., heptadecanoic acid, isoleucine, and methionine) whose catabolism includes the formation of propionyl-CoA, were found to inhibit ST and conidiospore pigment production. These results demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis. [ABSTRACT FROM AUTHOR]

The DNA damage response is a protective mechanism that ensures the maintenance of genomic integrity. We have used Aspergillus nidulans as a model system to characterize the DNA damage response caused by the antitopoisomerase I drug, camptothecin. We report the molecular characterization of a p34Cdc2-related gene, npkA, from A. nidulans. The npkA gene is transcriptionally induced by camptothecin and other DNA-damaging agents, and its induction in the presence of camptothecin is dependent on the uvsBATR gene. There were no growth defects, changes in developmental patterns, increased sensitivity to DNA-damaging agents, or effects on septation or growth rate in the A. nidulans npkA deletion strain. However, the ΔnpkA mutation can partially suppress HU sensitivity caused by the ΔuvsBATR and uvsD153ATRIP checkpoint mutations. We demonstrated that the A. nidulans uvsBATR gene is involved in DNA replication and the intra-S-phase checkpoints and that the ΔnpkA mutation can suppress its intra-S-phase checkpoint deficiency. There is a defect in both the intra-S-phase and DNA replication checkpoints due to the npkA inactivation when DNA replication is slowed at 6 mM HU. Our results suggest that the npkA gene plays a role in cell cycle progression during S-phase as well as in a DNA damage signal transduction pathway in A. nidulans. [ABSTRACT FROM AUTHOR]

We isolated the ganB gene encoding the Gα-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. [ABSTRACT FROM AUTHOR]

Unlike Pho85 of Saccharomyces cerevisiae, the highly related PHOA cyclin-dependent kinase (CDK) of Aspergillus nidulans plays no role in regulation of enzymes involved in phosphorous acquisition but instead modulates differentiation in response to environmental conditions, including limited phosphorous. Like PHO85, Aspergillus phoA is a nonessential gene. However, we find that expression of dominant-negative PHOA inhibits growth, suggesting it may have an essential but redundant function. Supporting this we have identified another cyclin-dependent kinase, PHOB, which is 77% identical to PHOA. Deletion of phoB causes no phenotype, even under phosphorous-limited growth conditions. To investigate the function of phoA/phoB, double mutants were selected from a cross of strains containing null alleles and by generating a temperature-sensitive allele of phoA in a ΔphoB background. Double-deleted ascospores were able to germinate but had a limited capacity for nuclear division, suggesting a cell cycle defect. Longer germination revealed morphological defects. The temperature-sensitive phoA allele caused both nuclear division and polarity defects at restrictive temperature, which could be complemented by expression of mammalian CDK5. Therefore, an essential function exists in A. nidulans for the Pho85-1ike kinase pair PHOA and PHOB, which may involve cell cycle control and morphogenesis. [ABSTRACT FROM AUTHOR]