Binuclear Cluster Research Articles

Transcriptome analysis reveals downregulation of virulence-associated genes expression in a low virulence Verticillium dahliae strain.

Verticillium dahliae causes wilt diseases and early senescence in numerous plants, including agricultural crops such as cotton. In this study, we studied two closely related V. dahliae strains, and found that V991w showed significantly reduced virulence on cotton than V991b. Comprehensive transcriptome analysis revealed various differentially expressed genes between the two strains, with more genes repressed in V991w. The downregulated genes in V991w were involved in production of hydrophobins, melanin, predicted aflatoxin, and membrane proteins, most of which are related to pathogenesis and multidrug resistance. Consistently, melanin production in V991w in vitro was compromised. We next obtained genomic variations between the two strains, demonstrating that transcription factor genes containing fungi specific transcription factor domain and fungal Zn2-Cys6 binuclear cluster domain were enriched in V991w, which might be related to pathogenicity-related genes downregulation. Thus, this study supports a model in which some virulence factors involved in V. dahliae pathogenicity were pre-expressed during in vitro growth before host interaction.

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q0 or Q0 H2 ). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q0 and O2 , or NADH and Q0 H2 respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.

Structural Evolution and Chemical Bonding in Bi-nuclear Niobium Sulfide Clusters: Nb2S n −/0 (n = 4–7)

Geometric and electronic structures of a series of bi-nuclear niobium sulfide clusters, Nb2S /0 (n = 4–7), were investigated by the theoretical calculations. Vertical detachment energies were estimated based on the generalized Koopmans’ theorem and were used to simulate the anionic photoelectron spectra. The structural evolution behaviors were found to be present in the sequential sulfidation of Nb2S −/0 . Concretely, the additional sulfur atoms tended to occupy the bridging sites of Nb2S /0 (n = 4, 5) clusters. Then varied poly-sulfur ligands (i.e., bridging S 2 2 , terminal S 2 2 and bridging S 3 2 ) began to emerge in the Nb2S /0 (n = 6, 7) clusters. Frontier molecular orbitals were analyzed to understand the chemical bonding. It was found that the highest occupied molecular orbital of Nb2S cluster, even the sulfur-rich Nb2S 7 cluster, still retained the Nb–Nb bonding features. The results were compared with the analogs Mo2S /0 (n = 4–7) clusters. The findings in structures and chemical bonding may provide insight into the properties of niobium sulfides, especially for the sulfur-rich surfaces.

Stb5p is involved in Kluyveromyces lactis response to 4-nitroquinoline-N-oxide stress.

In yeast, the STB5 gene encodes a transcriptional factor belonging to binuclear cluster class (Zn2Cys6) of transcriptional regulators specific to ascomycetes. In this study, we prepared the Kluyveromyces lactis stb5Δ strain and assessed its responses to different stresses. We showed that KlSTB5 gene is able to complement the deficiencies of Saccharomyces cerevisiae stb5Δ mutant. The results of phenotypic analysis suggested that KlSTB5 gene deletion did not sensitize K. lactis cells to oxidative stress inducing compounds but led to Klstb5Δ resistance to 4-nitroquinoline-N-oxide and hygromycin B. Expression analysis indicated that the loss of KlSTB5 gene function induced the transcription of drug efflux pump encoding genes that might contribute to increased 4-nitroquinoline-N-oxide and hygromycin B tolerance. Our results show that KlStb5p functions as negative regulator of some ABC transporter genes in K. lactis.

Three novel topologically different metal-organic frameworks built from 3-nitro-4-(pyridin-4-yl)benzoic acid.

The design and synthesis of metal-organic frameworks (MOFs) have attracted much interest due to the intriguing diversity of their architectures and topologies. However, building MOFs with different topological structures from the same ligand is still a challenge. Using 3-nitro-4-(pyridin-4-yl)benzoic acid (HL) as a new ligand, three novel MOFs, namely poly[[(N,N-dimethylformamide-κO)bis[μ2-3-nitro-4-(pyridin-4-yl)benzoato-κ3O,O':N]cadmium(II)] N,N-dimethylformamide monosolvate methanol monosolvate], {[Cd(C12H7N2O4)2(C3H7NO)]·C3H7NO·CH3OH}n, (1), poly[[(μ2-acetato-κ2O:O')[μ3-3-nitro-4-(pyridin-4-yl)benzoato-κ3O:O':N]bis[μ3-3-nitro-4-(pyridin-4-yl)benzoato-κ4O,O':O':N]dicadmium(II)] N,N-dimethylacetamide disolvate monohydrate], {[Cd2(C12H7N2O4)3(CH3CO2)]·2C4H9NO·H2O}n, (2), and catena-poly[[[diaquanickel(II)]-bis[μ2-3-nitro-4-(pyridin-4-yl)benzoato-κ2O:N]] N,N-dimethylacetamide disolvate], {[Ni(C12H7N2O4)2(H2O)2]·2C4H9NO}n, (3), have been prepared. Single-crystal structure analysis shows that the CdII atom in MOF (1) has a distorted pentagonal bipyramidal [CdN2O5] coordination geometry. The [CdN2O5] units as 4-connected nodes are interconnected by L- ligands to form a fourfold interpenetrating three-dimensional (3D) framework with a dia topology. In MOF (2), there are two crystallographically different CdII ions showing a distorted pentagonal bipyramidal [CdNO6] and a distorted octahedral [CdN2O4] coordination geometry, respectively. Two CdII ions are connected by three carboxylate groups to form a binuclear [Cd2(COO)3] cluster. Each binuclear cluster as a 6-connected node is further linked by acetate groups and L- ligands to produce a non-interpenetrating 3D framework with a pcu topology. MOF (3) contains two crystallographically distinct NiII ions on special positions. Each NiII ion adopts an elongated octahedral [NiN2O4] geometry. Each NiII ion as a 4-connected node is linked by L- ligands to generate a two-dimensional network with an sql topology, which is further stabilized by two types of intermolecular OW-HW...O hydrogen bonds to form a 3D supramolecular framework. MOFs (1)-(3) were also characterized by powder X-ray diffraction, IR spectroscopy and thermogravimetic analysis. Furthermore, the solid-state photoluminescence of HL and MOFs (1) and (2) have been investigated. The photoluminescence of MOFs (1) and (2) are enhanced and red-shifted with respect to free HL. The gas adsorption investigation of MOF (2) indicates a good separation selectivity (71) of CO2/N2 at 273 K (i.e. the amount of CO2 adsorption is 71 times higher than N2 at the same pressure).

Cu(ii)/Ni(ii)-organic frameworks constructed from the homometallic clusters by 5-(2-carboxyphenoxy)isophthalic acid and N-ligand: synthesis, structures and visible light-driven photocatalytic properties.

Four new complexes, namely, Cu2(O-cpia)(btb)0.5·(OH) (1), Cu3(O-cpia)2(bpy)2 (2), [Ni2(O-cpia)(phen)·(OH)·H2O]·2H2O (3) and [Ni3(O-cpia)2(bpy)3·2H2O]·2H2O (4) (O-cpia = 5-(2-carboxyphenoxy)isophthalic acid, btb = 1,4-bis(1,2,4-triazol-1-yl)butane, bpy = 4,4′-bipyridine) were successfully isolated under hydrothermal conditions. The four complexes exhibit different architectures constructed from different homometallic clusters varying from mononuclear, binuclear to tetranuclear metal(ii) polyhedra as Second Building Blocks (SBUs). 1 features a 3D framework constructed from the tetranuclear clusters [Cu4(μ3-OH)2] as SBUs, linked with Cu(1)O4N and Cu(2)O5 polyhedra by O-cpia/btb mixed linkers. 2 also exhibits a 3D structure based on trinuclear clusters [Cu3(COO)4] SBUs, bridged with Cu(1)O3N2 and Cu(2)O4 polyhedra via O-cpia/bpy mixed ligands. 3 shows a 2D network consisting of tetranuclear clusters [Ni4(μ3-OH)2] SBUs, which are bridged with Ni(1)O4N2 and Ni(2)O6 through O-cpia ligands. It is worth noting that 4, with a 3D structure, is generated from the binuclear clusters [Ni2(COO)4] (Ni(1)O4N) and mononuclear metal Ni(2) cores (Ni(2)O4N2) as SBUs, and bridged by O-cpia/bpy mixed ligands. Meanwhile, the degradation of dyes (RhB) by the complexes under visible light irradiation was studied. 1–4 are semiconducting in nature, with Eg of 1.30 eV (1), 1.78 eV (2), 2.85 eV (3) and 2.14 eV (4). Cu(ii) complexes 1 and 2 are highly efficient photocatalysts for the degradation of RhB under visible light irradiation.

Reactivation of the Ready and Unready Oxidized States of [NiFe]-Hydrogenases: Mechanistic Insights from DFT Calculations.

The apparently simple dihydrogen formation from protons and electrons (2H+ + 2e- ⇄ H2) is one of the most challenging reactions in nature. It is catalyzed by metalloenzymes of amazing complexity, called hydrogenases. A better understanding of the chemistry of these enzymes, especially that of the [NiFe]-hydrogenases subgroup, has important implications for production of H2 as alternative sustainable fuel. In this work, reactivation mechanism of the oxidized and inactive Ni-B and Ni-A states of the [NiFe]-hydrogenases active site has been investigated using density functional theory. Results obtained from this study show that one-electron reduction and protonation of the active site promote the removal of the bridging hydroxide ligand contained in Ni-B and Ni-A. However, this process is sufficient to activate only the Ni-B state. H2 binding to the active site is required to convert Ni-A to the active Ni-SIa state. Here, we also propose a reasonable structure for the spectroscopically well-characterized Ni-SIr and Ni-SU species, formed respectively from the one-electron reduction of Ni-B and Ni-A. Ni-SIr, depending on the pH at which the reaction occurs, features a bridging hydroxide ligand or a water molecule terminally coordinated to the Ni atom, whereas in Ni-SU a water molecule is terminally coordinated to the Fe atom, and the Cys64 residue is oxidized to sulfenate. The sulfenate oxygen atom in the Ni-A state affects the stereoelectronic properties of the binuclear cluster by modifying the coordination geometry of Ni, and consequently, by switching the regiochemistry of H2O and H2 binding from the Ni to the Fe atom. This effect is predicted to be at the origin of the different reactivation kinetics of the oxidized and inactive Ni-B and Ni-A states.

A potassium-encapsulated Preyssler-based structure modified by binuclear cyclic copper(II) clusters: photocatalytic and electrochemical properties

Through the use of a flexible ligand 1,1′-bis(3,5-dimethyl-1H-pyrazolate)methane (H2bdpm), a K-encapsulated Preyssler-based complex, [Cu4(H2bdpm)4(H2O)10(H6KP5W30O110)]·2(H2bdpm)·14H2O, has been synthesized under hydrothermal conditions. Single crystal X-ray diffraction analysis shows that the complex contains rare K-encapsulated Preyssler anions, which are linked by binuclear cyclic clusters [Cu2(H2bdpm)2]4+ to form a 1D chain. Adjacent chains are further connected by abundant intermolecular forces to construct a 2D supramolecular framework. The photocatalytic and electrochemical properties of the complex have been investigated. (1) The Preyssler-based POM-TMCs are rarely reported. (2) The K-encapsulated Preyssler anion is uncommonly observed in Preyssler-based compound. (3) Preyssler anions are linked by binuclear CuII clusters to form a 1D chain. (4) Compound 1 exhibits photocatalytic and electrochemical properties.

Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin

BackgroundFilamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol.ResultsA comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B.ConclusionHere we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.

Monocycle‐like Molecular Rotor Induces a Dielectric Relaxation and Dielectric Phase Transformation in an Organic–Inorganic Hybrid Supermolecule

A new strategy for the construction of crystalline supermolecular rotors is presented. We obtained the hybrid crystal [(18‐Crown‐6)K]3[NH2(CH3)2]2(Bi2Cl11)·2DMF (1) by introducing the flexible 18‐crown‐6 into the inorganic [Bi2Cl11]5– binuclear anion cluster lattice through the KCl (screw) to connect the organic molecule with inorganic component, in which the coordinated DMF (N,N‐Dimethylformamide) molecule functions as an polar rotator with monocycle shape, revealed by its significant dielectric relaxation above 358 K. The hybrid solid also exhibits dielectric phase transformation and conductivity switch around 413 K, the response mechanism was investigated by variable‐temperature X‐ray single crystal and powder diffraction as well as DSC, TG techniques. The analysis results reveal that the dielectric anomaly and conductivity switch are triggered by the losing of DMF molecules which is fixed with the [(18‐Crown‐6)K]+. And the ionic conductivity reach to 1.10 × 10–3 S cm–1 at 473 K. The interesting molecular structure and molecular thermal motion feature are promising application in the field of artificial molecular machine and dielectric switch.

Synthesis and magnetic properties of chiral molecular magnet regulated by terminal coordination groups

The solvothermal reaction of (HL = 2-(5-Pyrazinyl-1H-[1,2,4]triazol-3-yl)-pyrazine) and NiCl2·6H2O and Mn(HCOO)2·2H2O yielded two novel complexes, namely, Ni(L)(Cl)(DMF) (HPU-11) and {Mn(L)(HCOO)}n (HPU-12), which have been studied based on X-ray single crystal diffraction analysis. In HPU-11, two crystallographic independent NiII ions are connected by two L− ligands to generate a centrosymmetric binuclear cluster. By changing the terminal coordination atom Cl as HCOO, chiral magnet HPU-12 was obtained. In HPU-12, adjacent Mn ions are connected by HCOO− bridges giving rise to a one-dimensional chiral chain structure. The magnetic measurements show the presence of antiferromagnetic interactions between the magnetic centers of the two complexes.

Molecular basis of resistance to the microtubule-depolymerizing antitumor compound plocabulin

Plocabulin (PM060184) is a microtubule depolymerizing agent with potent antiproliferative activity undergoing phase II clinical trials for the treatment of solid tumors. Plocabulin shows antifungal activity virtually abolishing growth of the filamentous fungus Aspergillus nidulans. A. nidulans hyphae depend both on mitotic and interphase microtubules, as human cells. Here, we exploited the A. nidulans genetic amenability to gain insight into the mechanism of action of plocabulin. By combining mutations in the two A. nidulans β-tubulin isotypes we obtained a plocabulin-insensitive strain, showing that β-tubulin is the only molecular target of plocabulin in fungal cells. From a genetic screen, we recovered five mutants that show plocabulin resistance but do not carry mutations in β-tubulin. Resistance mutations resulted in amino acid substitutions in (1) two subunits of the eukaryotic translation initiation factor eIF2B activating the General Amino Acid Control, (2) TIM44, an essential component of the inner mitochondrial membrane translocase, (3) two transcription factors of the binuclear zinc cluster family potentially interfering with the uptake or efflux of plocabulin. Given the conservation of some of the identified proteins and their respective cellular functions in the tumor environment, our results pinpoint candidates to be tested as potential biomarkers for determination of drug efficiency.

Porous 10- and 12-vertex (bi)-p-dicarba-closo-boranedicarboxylates of cobalt and their gas adsorptive properties

Two new porous coordination polymers, [Co3(μ3-OH)(Bcb10DC)2.5(DEF)3(EtOH)1.5(H2O)0.5] · DEF · EtOH (3b) and [Co2(Bcb12DC)2(DMF)4(EtOH)] · 2EtOH (4) based on 10- and 12-vertex p-bicarboranedicarboxylic acids, 1,1′-bi-(1,10-dicarba-closo-decaborane)-10,10′-dicarboxylic acid (H2Bcb10DC) and 1,1′-bi-(1,12-dicarba-closo-decaborane)-12,12′-dicarboxylic acid (H2Bcb12DC) were synthesized. Their properties were compared with the two known analogues based on shorter ligand-homologues, [Co(cb10DC)(DMF)] (1) and [Co4(OH)2(cb12DC)3(DMF)2(H2O)4] · 3DMF · EtOH (2). The structure of 3b is based on a trinuclear cluster and has a 3D structure with a 5-c bnn underlying net, while 4 is based on a binuclear cluster and composed of stacked 2D sql layers. Activated 3′ with altered structure has SBET(N2) of 1012 m2 g−1 and 4′ with mostly retained structure 920 m2 g−1. Gas adsorption by 3′ and 4′ at 1 bar is: 80.1 cm3 g-1 (0.72%wt) and 81.3 cm3 g-1 (0.73%wt) H2 at 77 K; 16.7 and 21.9 cm3 g-1 CO2 at 273 K; 6.2 and 10.0 cm3 g-1 of CH4 at 273 K. The isosteric heat of adsorption Qst at zero coverage are respectively: 5.0 and 4.5 kJ mol−1 for H2, 16.7 and 21.9 for CO2; 6.2 and 10.0 for CH4. 2′ with ∼645 m2 g−1 (calc. 883 m2g−1) has the highest mass-specific H2 adsorption, >30% than other compounds, while Qst is close to 1′ and slightly higher than for 3′ and 4´. Adsorption of CO2 and CH4 and Qst are ∼1.5–2 times lower for 3′ and 4′ compared to 1′ and 2´. The surface area specific adsorption of H2, CO2 and CH4 is the highest for 1´. The CO2/CH4 IAST selectivities (0.15/0.85; 1 bar, 298 K) are in 2–4.5 range. 3′ and 4′ retain a significant part of porosity after soaking in water unlike 1′ and 2´.

Crystal structures of manganese-dependent transcriptional repressor MntR (Rv2788) from Mycobacterium tuberculosis in apo and manganese bound forms

The pathogenic Mycobacterium tuberculosis encodes two members of the DtxR family metalloregulators, IdeR and MntR. IdeR represses gene expression in response to ferrous iron, while MntR (Rv2788) functions as a manganese-dependent transcriptional repressor, which represses the expression of manganese transporter genes to maintain manganese homeostasis. Although the structural study towards IdeR is in-depth, there is no MntR structure available. Herein, we report both apo and manganese bound forms of MntR structures from M. tuberculosis. MntR has evolved into two metal ion binding sites like other DtxR proteins and for the first time, we captured the two sites fully occupied by its natural ions with one Mn2+ ion at the first site and two Mn2+ ions at the second binding site (binuclear manganese cluster). The conformation change of MntR resulting from manganese binding could prime the MntR for DNA binding, which is a conserved activation mechanism among DtxR family.

Interplay between CN- Ligands and the Secondary Coordination Sphere of the H-Cluster in [FeFe]-Hydrogenases.

The catalytic cofactor of [FeFe]-hydrogenses (H-cluster) is composed of a generic cubane [4Fe-4S]-cluster (4FeH) linked to a binuclear iron-sulfur cluster (2FeH) that has an open coordination site at which the reversible conversion of protons to molecular hydrogen occurs. The (2FeH) subsite features a diatomic coordination sphere composed of three CO and two CN- ligands affecting its redox properties and providing excellent probes for FTIR spectroscopy. The CO stretch vibrations are very sensitive to the redox changes within the H-cluster occurring during the catalytic cycle, whereas the CN- signals seem to be relatively inert to these effects. This could be due to the more structural role of the CN- ligands tightly anchoring the (2FeH) unit to the protein environment through hydrogen bonding. In this work we explore the effects of structural changes within the secondary ligand sphere affecting the CN- ligands on FTIR spectroscopy and catalysis. By comparing the FTIR spectra of wild-type enzyme and two mutagenesis variants, we are able to assign the IR signals of the individual CN- ligands of the (2FeH) site for different redox states of the H-cluster. Moreover, protein film electrochemistry reveals that targeted manipulation of the secondary coordination sphere of the proximal CN- ligand (i.e., closest to the (4FeH) site) can affect the catalytic bias. These findings highlight the importance of the protein environment for re-adjusting the catalytic features of the H-cluster in individual enzymes and provide valuable information for the design of artificial hydrogenase mimics.

Structures and properties of five metal–organic frameworks based on 3,3′,5,5′-azoxybenzenetetracarboxylic acid and different secondary building units

Abstract Five new metal–organic frameworks (MOFs), named [Zn 2 (aobtc)(H 2 O) 4 ] ( 1 ), [Mn 3 (Haobtc) 2 (DMA) 2 (H 2 O) 2 ]·2H 2 O ( 2 ), [Co 3 (Haobtc) 2 (H 2 O) 4 ]·11H 2 O ( 3 ), [Co 3 (aobtc) 2 ]·Himi·H 3 O·3H 2 O ( 4 ), [Cd 2 (aobtc)(bpy)]·4.5H 2 O ( 5 ) (H 4 aobtc = 3,3′,5,5′-azoxybenzenetetracarboxylic acid, bpy = 4,4′-bipyridine, imi = imidazole), have been designed and syntheses. Their structures were determined by single-crystal X-ray diffraction analyses and further characterized by elemental analyses, IR spectra and TG analyses. The five compounds exhibit diverse secondary building units (SBUs) and show various structures with different porosity. Compound 1 is a two-dimensional (2D) layer with (4,4)- sql topology based on binuclear Zn cluster (SBU). Both compounds 2 and 3 possess 2D framework with (3,6)-connected kgd topology, based on trinuclear Mn and Co clusters, respectively. Compound 4 shows a 3D framework with (3,8)-connected flu topology, containing linear trinuclear Co clusters as 8-connected nodes. The 3D framework of Compound 5 is built from infinite Cd–O–Cd rod SBUs. The effects of stacking modes on the crystal architectures are discussed. The solid-state photoluminescence for 1 and 5 , the magnetic properties for 2 – 4 , and CO 2 adsorption for 3 were also investigated.

Metal-to-metal charge-transfer transitions in Prussian blue hexacyanochromate analogues

Time-dependent density functional theory (TD-DFT) calculations are presented for a series of Prussian blue analogues (PBAs) based on hexacyanochromate in order to investigate optical metal-to-metal charge-transfer (MMCT) transitions. PBE0 hybrid DFT calculations at fixed geometry were carried out on binuclear units of (CN)5-MA-NC-CrIII-(CN)5, with MA=VII, VIII, and CrII. The MMCT transitions occurred at 743nm for the VII-CrIII PBA and at 780nm for the VIII-CrIII PBA. The ordering of the transition energy and strength for these analogues agreed with data available in the literature. The transition for the CrII-CrIII MMCT was found to occur at 284nm. This study shows that TD-DFT is a powerful method to study optical properties of PBAs and that the results for the relatively simple binuclear cluster are comparable to solid PBAs, where MMCT transitions are typically the dominant feature in the visible part of the absorption spectrum.

Network of nutrient-sensing pathways and a conserved kinase cascade integrate osmolarity and carbon sensing in Neurospora crassa

Identifying nutrients available in the environment and utilizing them in the most efficient manner is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of carbohydrates, from simple sugars to the complex carbohydrates found in plant cell walls. The zinc binuclear cluster transcription factor CLR-1 is necessary for utilization of cellulose, a major, recalcitrant component of the plant cell wall; however, expression of clr-1 in the absence of an inducer is not sufficient to induce cellulase gene expression. We performed a screen for unidentified actors in the cellulose-response pathway and identified a gene encoding a hypothetical protein (clr-3) that is required for repression of CLR-1 activity in the absence of an inducer. Using clr-3 mutants, we implicated the hyperosmotic-response pathway in the tunable regulation of glycosyl hydrolase production in response to changes in osmolarity. The role of the hyperosmotic-response pathway in nutrient sensing may indicate that cells use osmolarity as a proxy for the presence of free sugar in their environment. These signaling pathways form a nutrient-sensing network that allows Ncrassa cells to tightly regulate gene expression in response to environmental conditions.

Regulatory Mechanisms of a Highly Pectinolytic Mutant of Penicillium occitanis and Functional Analysis of a Candidate Gene in the Plant Pathogen Fusarium oxysporum.

Penicillium occitanis is a model system for enzymatic regulation. A mutant strain exhibiting constitutive overproduction of different pectinolytic enzymes both under inducing (pectin) or repressing conditions (glucose) was previously isolated after chemical mutagenesis. In order to identify the molecular basis of this regulatory mechanism, the genomes of the wild type and the derived mutant strain were sequenced and compared, providing the first reference genome for this species. We used a phylogenomic approach to compare P. occitanis with other pectinolytic fungi and to trace expansions of gene families involved in carbohydrate degradation. Genome comparison between wild type and mutant identified seven mutations associated with predicted proteins. The most likely candidate was a mutation in a highly conserved serine residue of a conserved fungal protein containing a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain and a fungus-specific transcription factor regulatory middle homology region. To functionally characterize the role of this candidate gene, the mutation was recapitulated in the predicted orthologue Fusarium oxysporum, a vascular wilt pathogen which secretes a wide array of plant cell wall degrading enzymes, including polygalacturonases, pectate lyases, xylanases and proteases, all of which contribute to infection. However, neither the null mutant nor a mutant carrying the analogous point mutation exhibited a deregulation of pectinolytic enzymes. The availability, annotation and phylogenomic analysis of the P. occitanis genome sequence represents an important resource for understanding the evolution and biology of this species, and sets the basis for the discovery of new genes of biotechnological interest for the degradation of complex polysaccharides.

Functional Profiling of Transcription Factor Genes in Neurospora crassa.

Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa. We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6) binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed), followed by asexual sporulation (38%), and the various stages of sexual development (19%). Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated.