A type II phosphatidylinositol-4-kinase coordinates sorting of cargo polarizing by endocytic recycling.

Depending on their phosphorylation status, derivatives of phosphatidylinositol play important roles in vesicle identity, recognition and intracellular trafficking processes. In eukaryotic cells, phosphatidylinositol-4 phosphate pools generated by specific kinases are key determinants of the conventional secretion pathways. Earlier work in yeast has classified phosphatidylinositol-4 kinases in two types, Stt4p and Pik1p belonging to type III and Lsb6p to type II, with distinct cellular localizations and functions. Eurotiomycetes appear to lack Pik1p homologues. In Aspergillus nidulans, unlike homologues in other fungi, AnLsb6 is associated to late Golgi membranes and when heterologously overexpressed, it compensates for the thermosensitive phenotype in a Saccharomyces cerevisiae pik1 mutant, whereas its depletion leads to disorganization of Golgi-associated PHOSBP-labelled membranes, that tend to aggregate dependent on functional Rab5 GTPases. Evidence provided herein, indicates that the single type II phosphatidylinositol-4 kinase AnLsb6 is the main contributor for decorating secretory vesicles with relevant phosphatidylinositol-phosphate species, which navigate essential cargoes following the route of apical polarization via endocytic recycling.

Modeling Studies of the Mechanism of Context-Dependent Bidirectional Movements of Kinesin-14 Motors.

Kinesin-14s, a subfamily of the large superfamily of kinesin motor proteins, function mainly in spindle assembly and maintenance during mitosis and meiosis. KlpA from Aspergillus nidulans and GiKIN14a from Giardia intestinalis are two types of kinesin-14s. Available experimental results puzzlingly showed that while KlpA moves preferentially toward the minus end in microtubule-gliding setups and inside parallel microtubule overlaps, it moves preferentially toward the plus end on single microtubules. More puzzlingly, the insertion of an extra polypeptide linker in the central region of the neck stalk switches the motility direction of KlpA on single microtubules to the minus end. Prior experimental results showed that GiKIN14a moves preferentially toward the minus end on single microtubules in either tailless or full-length forms. The tail not only greatly enhances the processivity but also accelerates the ATPase rate and velocity of GiKIN14a. The insertion of an extra polypeptide linker in the central region of the neck stalk reduces the ATPase rate of GiKIN14a. However, the underlying mechanism of these puzzling dynamical features for KlpA and GiKIN14a is unclear. Here, to understand this mechanism, the dynamics of KlpA and GiKIN14a were studied theoretically on the basis of the proposed model, incorporating potential changes between the kinesin head and microtubule, as well as the potential between the tail and microtubule. The theoretical results quantitatively explain the available experimental results and provide predicted results. It was found that the elasticity of the neck stalk determines the directionality of KlpA on single microtubules and affects the ATPase rate and velocity of GiKIN14a on single microtubules.

Heterologous production of bioactive xenoacremone analogs in Aspergillus nidulans.

Tyrosine-decahydrofluorene derivatives are a class of hybrid compounds that integrate the properties of polyketides and nonribosomal peptides. These compounds feature a [6.5.6] tricarbocyclic core and a para-cyclophane ether moiety in their structures and exhibit anti-tumor and anti-microbial activities. In this study, we constructed the biosynthetic pathway of xenoacremones from Xenoacremonium sinensis ML-31 in the Aspergillus nidulans host, resulting in the identification of four novel tyrosine-decahydrofluorene analogs, xenoacremones I-L (1-4), along with two known analogs, xenoacremones A and B. Remarkably, compounds 3 and 4 contained a 12-membered para-cyclophane ring system, which is unprecedented among tyrosine-decahydrofluorene analogs in X. sinensis. The successful reconstruction of the biosynthetic pathway and the discovery of novel analogs demonstrate the utility of heterologous expression strategy for the generation of structurally diverse natural products with potential biological activities.

The role of peroxiredoxins PrxA and PrxB in the antioxidant response, carbon utilization and development in Aspergillus nidulans.

In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.

Urease and Carbonic Anhydrase Inhibitory Effect of Xanthones from Aspergillus nidulans, an Endophytic Fungus of Nyctanthes arbor-tristis.

Urease plays a major role in the pathogenesis of peptic and gastric ulcer and also causes acute pyelonephritis and development of infection-induced reactive arthritis. Carbonic anhydrases (CA) cause pathological disorders such as epilepsy (CA I), glaucoma, gastritis, renal, pancreatic carcinomas, and malignant brain tumors (CA II). Although various synthetic urease and carbonic anhydrase inhibitors are known, these have many side effects. Hence, present studies were undertaken on ethyl acetate extract of Aspergillus nidulans, an endophytic fungus separated from the leaves of Nyctanthes arbor-tristis Linn. and led to the isolation of five furanoxanthones, sterigmatin (1: ), sterigmatocystin (3: ), dihydrosterigmatocystin (4: ), oxisterigmatocystin C (5: ), acyl-hemiacetal sterigmatocystin (6: ), and a pyranoxanthone (2: ). Acetylation of 3: gave compound O-acetyl sterigmatocystin (7: ). Their chemical structures were elucidated by 1H and 13C NMR and MS. The inhibitory effect of isolated compounds was evaluated on urease and carbonic anhydrase (bCA II) enzymes in vitro. Compounds 3: and 6: showed significant urease inhibition (IC50 19 and 21 µM), while other compounds exhibited varying degrees of urease inhibition (IC50 33 - 51 µM). Compounds 4, 6: and 7: exhibited significant inhibition of bCA II (IC50 values 21, 25 and 18 µM respectively), compounds 1: -3: displayed moderate inhibition (IC50 61, 76 and 31 µM respectively) while 5: showed no inhibition. A mechanistic study of the most active urease inhibitors was also performed using enzyme kinetics and molecular docking. All compounds were found non-toxic on the NIH-3T3 cell line.

Mining an O-methyltransferase for de novo biosynthesis of physcion in Aspergillus nidulans.

Physcion is one of natural anthraquinones, registered as a novel plant-derived fungicide due to its excellent prevention of plant disease. However, the current production of physcion via plant extraction limits its yield promotion and application. Here, a pair of polyketide synthases (PKS) in emodin biosynthesis were used as probes to mining the potential O-methyltransferase (OMT) responsible for physcion biosynthesis. Further refinement using the phylogenetic analysis of the mined OMTs revealed a distinct OMT (AcOMT) with the ability of transferring a methyl group to C-6 hydroxyl of emodin to form physcion. Through introducing AcOMT, we successfully obtained the de novo production of physcion in Aspergillus nidulans. The physcion biosynthetic pathway was further rationally engineered by expressing the decarboxylase genes from different fungi. Finally, the titer of physcion reached to 64.6 mg/L in shake-flask fermentation through enhancing S-adenosylmethionine supply. Our work provides a native O-methyltransferase for physcion biosynthesis and lays the foundation for further improving the production of physcion via a sustainable route. KEY POINTS: • Genome mining of the native O-methyltransferase responsible for physcion biosynthesis • De novo biosynthesis of physcion in the engineered Aspergillus nidulans • Providing an alternative way to produce plant-derived fungicide physcion.

Kinesin-1 autoinhibition facilitates the initiation of dynein cargo transport.

The functional significance of Kinesin-1 autoinhibition has been unclear. Kinesin-1 transports multiple cargoes including cytoplasmic dynein to microtubule plus ends. From a genetic screen for Aspergillus mutants defective in dynein-mediated early endosome transport, we identified a kinesin-1 mutation kinAK895* at the C-terminal IAK motif involved in autoinhibition. The kinA∆IAK and kinAK895E mutants exhibited a similar defect in dynein-mediated early endosome transport, verifying the importance of kinesin-1 autoinhibition in dynein-mediated transport. Kinesin-1 autoinhibition is not critical for dynein accumulation at microtubule plus ends or for the secretory vesicle cargoes of kinesin-1 to reach the hyphal tip. However, it facilitates dynein to initiate early endosome transport. This is unrelated to a direct competition between dynein and kinesin-1 on early endosomes because kinesin-3 rather than kinesin-1 drives the plus-end-directed early endosome movement. This effect of kinesin-1 autoinhibition on dynein-mediated early endosome transport is related to cargo adapter-mediated dynein activation but at a step beyond the switching of dynein from its autoinhibited conformation.

Asperemestrins A-D, Emestrin Hybrid Polymers with Bridged Skeletons from the Endophytic Fungus Aspergillus nidulans.

Four emestrin hybrid polymers, asperemestrins A-D (1-4, respectively), were isolated from the fungus Aspergillus nidulans. Asperemestrins A-C are the first examples of emestrin-sterigmatocystin heterodimers bearing a 7/5/6/6/5/5/6/6/6 nonacyclic system with a 2,5-diazabicyclo[2.2.2]octane-3,6-dione core, while asperemestrin D features an unprecedented 2,15-dithia-17,19-diazabicyclo[14.2.2]icosa-4,8-diene-12,18,20-trione core skeleton. Their structures were determined by extensive spectroscopic data, electronic circular dichroism calculations, and single-crystal X-ray diffraction. Asperemestrin B showed moderate cytotoxicity against cancer cell lines, including SU-DHL-2, HEPG2, and HL-60.

Antibacterial diphenyl ether production induced by co-culture of Aspergillus nidulans and Aspergillus fumigatus.

Fungi are a rich source of secondary metabolites with potent biological activities. Co-culturing a fungus with another microorganism has drawn much attention as a practical method for stimulating fungal secondary metabolism. However, in most cases, the molecular mechanisms underlying the activation of secondary metabolite production in co-culture are poorly understood. To elucidate such a mechanism, in this study, we established a model fungal-fungal co-culture system, composed of Aspergillus nidulans and Aspergillus fumigatus. In the co-culture of A. nidulans and A. fumigatus, production of antibacterial diphenyl ethers was enhanced. Transcriptome analysis by RNA-sequencing showed that the co-culture activated expression of siderophore biosynthesis genes in A. fumigatus and two polyketide biosynthetic gene clusters (the ors and cic clusters) in A. nidulans. Gene disruption experiments revealed that the ors cluster is responsible for diphenyl ether production in the co-culture. Interestingly, the ors cluster was previously reported to be upregulated by co-culture of A. nidulans with the bacterium Streptomyces rapamycinicus; orsellinic acid was the main product of the cluster in that co-culture. In other words, the main product of the ors cluster was different in fungal-fungal and bacterial-fungal co-culture. The genes responsible for biosynthesis of the bacterial- and fungal-induced polyketides were deduced using a heterologous expression system in Aspergillus oryzae. The molecular genetic mechanisms that trigger biosynthesis of two different types of compounds in A. nidulans in response to the fungus and the bacterium were demonstrated, which provides an insight into complex secondary metabolic response of fungi to microorganisms. KEY POINTS: • Co-culture of two fungal species triggered antibiotic diphenyl ether production. • The co-culture affected expression levels of several genes for secondary metabolism. • Gene cluster essential for induction of the antibiotics production was determined.

Induction of Iron Stress in Hepatocellular Carcinoma Cell Lines by Siderophore of Aspergillus nidulans Towards Promising Anticancer Effect.

Hepatocellular carcinoma is among the leading causes of cancer-related deaths worldwide and needs efficient and feasible approach of treatment. Present study focuses on exploring the anticancer activity of a secondary metabolite called siderophore of Aspergillus nidulans against hepatocellular carcinoma cell line HepG2. These small peptides are produced by microorganisms including fungi for scavenging iron from its surroundings. Fungi including Aspergillus spp. are known to produce siderophores under iron-limited conditions. Siderophores have high affinity towards iron and are classified into various types. In the present study, siderophore isolated and purified from fungal cultures was confirmed to be of hydroxamate type by chrome azurol sulfonate and Atkin's assay. HPLC analysis confirmed purity while LC-ESI-MS revealed that the siderophore is triacetyl fusigen. Cancerous cells, HepG2, grown under siderophore treatment showed inhibition in growth and proliferation in a dose- and time-dependent manner. Reduction in viability and metabolic activity was evident upon treatment as seen in trypan blue, MTT and WST assay. Fluorescent staining using PI and DAPI confirmed the same while DCFDA staining revealed increased reactive oxygen species production which might have led to cell death and deterioration. Such increase in ROS has been correlated with iron accumulation by assessing intracellular iron level through ICP-MS. To assess the effect of siderophore treatment on normal cells, WRL-68, same assays were carried out but the effect was mostly non-significant up to 48 h. Thus, present work suggests that an optimum dose of siderophore purified from A. nidulans culture might prove a useful anticancer agent.

Optimizing microtubule arrangements for rapid cargo capture.

Cellular functions such as autophagy, cell signaling, and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow undirected movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus ends relative to the initial cargo position. We use analytic mean first-passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule arrangements that minimize mean cargo capture times. Namely, we show that configurations with a single microtubule plus end abutting the distal tip and broadly distributed other plus ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between the distribution of microtubule tips and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

The DUG Pathway Governs Degradation of Intracellular Glutathione in Aspergillus nidulans.

Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance for deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigated functions of the dugB and dugC genes in the model fungus Aspergillus nidulans These genes are orthologues of dug2 and dug3, which are involved in cytosolic GSH degradation in Saccharomyces cerevisiae The deletion of dugB, dugC, or both resulted in a moderate increase in the GSH content in mycelia grown on glucose, reduced conidium production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding mitogen-activated protein (MAP) kinase pathway elements (e.g., steC, sskB, hogA, and mkkA) or regulatory proteins of conidiogenesis and sexual differentiation (e.g., flbA, flbC, flbE, nosA, rosA, nsdC, and nsdD) were downregulated in the ΔdugB ΔdugC mutant. Deletion of dugB and/or dugC slowed the depletion of GSH pools during carbon starvation. It also reduced accumulation of reactive oxygen species and decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions-in contrast to mycotoxin production-were controlled at the posttranscriptional level. We suggest that GSH connects starvation and redox regulation to each other: cells utilize GSH as a stored carbon source during starvation. The reduction of GSH content alters the redox state, activating regulatory pathways responsible for carbon starvation stress responses.IMPORTANCE Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character, and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, and secondary metabolite production (including mycotoxin formation), as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH-producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity, or oxidative stress tolerance of a fungal strain is manipulated.

PxdA interacts with the DipA phosphatase to regulate peroxisome hitchhiking on early endosomes.

In canonical microtubule-based transport, adaptor proteins link cargoes to dynein and kinesin motors. Recently, an alternative mode of transport known as "hitchhiking" was discovered, where cargoes achieve motility by hitching a ride on already-motile cargoes, rather than attaching to a motor protein. Hitchhiking has been best studied in two filamentous fungi, Aspergillus nidulans and Ustilago maydis. In U. maydis, ribonucleoprotein complexes, peroxisomes, lipid droplets (LDs), and endoplasmic reticulum hitchhike on early endosomes (EEs). In A. nidulans, peroxisomes hitchhike using a putative molecular linker, peroxisome distribution mutant A (PxdA), which associates with EEs. However, whether other organelles use PxdA to hitchhike on EEs is unclear, as are the molecular mechanisms that regulate hitchhiking. Here we find that the proper distribution of LDs, mitochondria, and preautophagosomes do not require PxdA, suggesting that PxdA is a peroxisome-specific molecular linker. We identify two new pxdA alleles, including a point mutation (R2044P) that disrupts PxdA's ability to associate with EEs and reduces peroxisome movement. We also identify a novel regulator of peroxisome hitchhiking, the phosphatase DipA. DipA colocalizes with EEs and its association with EEs relies on PxdA. Together, our data suggest that PxdA and the DipA phosphatase are specific regulators of peroxisome hitchhiking on EEs.

Internalization of the Aspergillus nidulans AstA Transporter into Mitochondria Depends on Growth Conditions, and Affects ATP Levels and Sulfite Oxidase Activity.

The astA gene encoding an alternative sulfate transporter was originally cloned from the genome of the Japanese Aspergillus nidulans isolate as a suppressor of sulfate permease-deficient strains. Expression of the astA gene is under the control of the sulfur metabolite repression system. The encoded protein transports sulfate across the cell membrane. In this study we show that AstA, having orthologs in numerous pathogenic or endophytic fungi, has a second function and, depending on growth conditions, can be translocated into mitochondria. This effect is especially pronounced when an astA-overexpressing strain grows on solid medium at 37 °C. AstA is also recruited to the mitochondria in the presence of mitochondria-affecting compounds such as menadione or antimycin A, which are also detrimental to the growth of the astA-overexpressing strain. Disruption of the Hsp70-Porin1 mitochondrial import system either by methylene blue, an Hsp70 inhibitor, or by deletion of the porin1-encoding gene abolishes AstA translocation into the mitochondria. Furthermore, we observed altered ATP levels and sulfite oxidase activity in the astA-overexpressing strain in a manner dependent on sulfur sources. The presented data indicate that AstA is also involved in the mitochondrial sulfur metabolism in some fungi, and thereby indirectly manages redox potential and energy state.

Tolerance to alkaline ambient pH in Aspergillus nidulans depends on the activity of ENA proteins.

Tolerance of microorganisms to abiotic stress is enabled by regulatory mechanisms that coordinate the expression and activity of resistance genes. Alkalinity and high salt concentrations are major environmental physicochemical stresses. Here, we analyzed the roles of sodium-extrusion family (ENA) transporters EnaA, EnaB and EnaC in the response to these stress conditions in the filamentous fungus Aspergillus nidulans. While EnaC has a minor role, EnaB is a key element for tolerance to Na+ and Li+ toxicity. Adaptation to alkaline pH requires the concerted action of EnaB with EnaA. Accordingly, expression of enaA and enaB was induced by Na+, Li+ and pH 8. These expression patterns are altered in a sltAΔ background and completely inhibited in a mutant expressing non-functional PacC protein (palH72). However, a constitutively active PacC form was not sufficient to restore maximum enaA expression. In agreement with their predicted role as membrane ATPases, EnaA localized to the plasma membrane while EnaB accumulated at structures resembling the endoplasmic reticulum. Overall, results suggest different PacC- and SltA-dependent roles for EnaB in pH and salt homeostasis, acting in coordination with EnaA at pH 8 but independently under salt stress.

The splicing-factor Prp40 affects dynein-dynactin function in Aspergillus nidulans.

The multi-component cytoplasmic dynein transports cellular cargoes with the help of another multi-component complex dynactin, but we do not know enough about factors that may affect the assembly and functions of these proteins. From a genetic screen for mutations affecting early-endosome distribution in Aspergillus nidulans, we identified the prp40AL438* mutation in Prp40A, a homologue of Prp40, an essential RNA-splicing factor in the budding yeast. Prp40A is not essential for splicing, although it associates with the nuclear splicing machinery. The prp40AL438* mutant is much healthier than the ∆prp40A mutant, but both mutants exhibit similar defects in dynein-mediated early-endosome transport and nuclear distribution. In the prp40AL438* mutant, the frequency but not the speed of dynein-mediated early-endosome transport is decreased, which correlates with a decrease in the microtubule plus-end accumulations of dynein and dynactin. Within the dynactin complex, the actin-related protein Arp1 forms a mini-filament. In a pull-down assay, the amount of Arp1 pulled down with its pointed-end protein Arp11 is lowered in the prp40AL438* mutant. In addition, we found from published interactome data that a mammalian Prp40 homologue PRPF40A interacts with Arp1. Thus, Prp40 homologues may regulate the assembly or function of dynein-dynactin and their mechanisms deserve to be further studied.

Epoxidation Catalyzed by the Nonheme Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, AsqJ: Mechanistic Elucidation of Oxygen Atom Transfer by a Ferryl Intermediate.

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.

Siderophore-assisted cadmium hyperaccumulation in Bacillus subtilis.

Siderophores (Gk iron carriers) are low molecular weight secondary metabolites produced by bacteria, fungi, and plants that have strong binding affinity for iron. Owing to their iron-chelating ability, they are produced mainly when the organism faces iron scarcity. The present study empirically investigated the importance of applying hydroxamate siderophore extracted from Aspergillus nidulans to the cells of Bacillus subtilis for bioremediation of cadmium salt. This investigation deals with siderophore-mediated intracellular Cd accumulation by bacterial cells, growth estimation, biochemical assays like lipid peroxidation, total protein content, carbohydrate content, and iron content estimation. In silico docking and STRING analyses revealed specific interaction between Aspergillus siderophore and receptors present on B. subtilis. Estimation of intracellular Cd by atomic absorption spectroscopy showed more accumulation of Cd ions by B. subtilis in the presence of hydroxamate siderophore. This suggests a possibility of confiscating environmental Cd2+ by utilizing metal chelation property of siderophores and hence can lead to emerging bioremediation mechanisms for heavy metals. In silico studies support experimental investigation and suggest higher affinity of siderophore for Cd ions as compared with ferric ions.

LIS1 regulates cargo-adapter-mediated activation of dynein by overcoming its autoinhibition in vivo.

Deficiency of the LIS1 protein causes lissencephaly, a brain developmental disorder. Although LIS1 binds the microtubule motor cytoplasmic dynein and has been linked to dynein function in many experimental systems, its mechanism of action remains unclear. Here, we revealed its function in cargo-adapter-mediated dynein activation in the model organism Aspergillus nidulans Specifically, we found that overexpressed cargo adapter HookA (Hook in A. nidulans) missing its cargo-binding domain (ΔC-HookA) causes dynein and its regulator dynactin to relocate from the microtubule plus ends to the minus ends, and this relocation requires LIS1 and its binding protein, NudE. Astonishingly, the requirement for LIS1 or NudE can be bypassed to a significant extent by mutations that prohibit dynein from forming an autoinhibited conformation in which the motor domains of the dynein dimer are held close together. Our results suggest a novel mechanism of LIS1 action that promotes the switch of dynein from the autoinhibited state to an open state to facilitate dynein activation.

Overexpression of an LaeA-like Methyltransferase Upregulates Secondary Metabolite Production in Aspergillus nidulans.

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA (mcrAΔ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG, a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrAΔ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrAΔ generally resulted in greater upregulation of SMs than upregulation of llmG, indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG. However, the combination of mcrAΔ and upregulation of llmG generally resulted in greater compound production than mcrAΔ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.