Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Conditional lethality involving a cytoplasmic mutant and chlorophyll-deficient malate dehydrogenase mutants in soybean

Summary Conditional lethality in soybean, Glycine max (L.) Merr., occurred in F₂ plants when cytoplasmicchlorophyll mutant Genetic Type T275 was the female parent and when either nuclear mutants T253 or T323 plants were the male parents. Mutant T253 [Mdh1-n (Urbana) y20 (Urbana) k2] is missing two of three mitochondrial malate dehydrogenase isozymes [Mdh1-n (Urbana)] and has yellowish-green leaves [y20 (Urbana)] and a tan-saddle pattern seed coat (k2). Mutant T323 [Mdh1-n (Ames 2) y20 (Ames 2)] also is missing two of three mitochondrial malate dehydrogenase isozymes [Mdh1-n (Ames 2)] and has yellowishgreen leaves [y20 (Ames 2)], but has yellow seed coat (K2). Mutants T275, T253, and T323 are viable both in the field and glasshouse. The genotypes cyt-Y2 Mdh1-n (Urbana) y20 (Urbana) k2/Mdh1-n (Urbana) y20 (Urbana) k2 and cyt-Y2 Mdh1-n (Ames 2) y20 (Ames 2)/Mdh1-n (Ames 2) y20 (Ames 2) are conditional lethals. These genotypes are lethal under field conditions, but plants survive in reduced light under shadecloth in the glasshouse. We do not know if their interaction with cyt-Y2 is due to Mdh1-n, y20, or Mdh1-n y20. The reciprocal cross (cyt-Y2 as male parent) gives viable genotypes. These conditional lethal genotypes should be useful for studies on the interaction between organelle and nuclear genomes.This is journal paper no. J-14777 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011-1010. Project 2985     

Synthesis and Analysis of Nucleosides Bearing Pyrrolepolyamide Binding to DNA

Abstract

An efficient synthesis of adenosine bearing pyrrolepolyamide 1 was achieved by coupling of 3 with 2. The CD spectra obtained at several [ligand ]/[duplex] ratios allowed verification of the formation complex of the DNA duplex [d(CGCAAATTGGC)/d(GCCAATTTGCG)] with 1.     

Constructing more informative plant–pollinator networks: visitation and pollen deposition networks in a heathland plant community

Interaction networks are widely used as tools to understand plant–pollinator communities, and to examine potential threats to plant diversity and food security if the ecosystem service provided by pollinating animals declines. However, most networks to date are based on recording visits to flowers, rather than recording clearly defined effective pollination events. Here we provide the first networks that explicitly incorporate measures of pollinator effectiveness (PE) from pollen deposition on stigmas per visit, and pollinator importance (PI) as the product of PE and visit frequency. These more informative networks, here produced for a low diversity heathland habitat, reveal that plant–pollinator interactions are more specialized than shown in most previous studies. At the studied site, the specialization index was lower for the visitation network than the PE network, which was in turn lower than for the PI network. Our study shows that collecting PE data is feasible for community-level studies in low diversity communities and that including information about PE can change the structure of interaction networks. This could have important consequences for our understanding of threats to pollination systems.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Study of the binding of jatrophone to Escherichia coli s-ribonucleic acid

The interaction of jatrophone with sRNA from Escherichia coli has been investigated through UV, CD, and 1H NMR measurements. The results obtained show that the interaction with jatrophone increases the stability of the polynucleotide. It appears that the optical properties of jatrophone depend upon the jatrophone/nucleotide ratio. The observed behaviour can only be explained by the existence of different types of interaction between jatrophone and sRNA. Even for a jatrophone/nucleotide ratio as low as 0.17 the 1H NMR spectra show a multiplicity of resonances that can only be explained by the simultaneous existence of two different binding modes involving the jatrophone molecules.     

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide γ-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10–40°C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10°C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.     

Nutritional interaction in an alga-barnacle association

Nitrogen is the major growth-limiting nutrient for marine algae. One potential source of nitrogen for marine algae is ammonium released by invertebrates. Many mid-intertidal reefs in northeastern New Zealand are dominated by a close association between the honeycomb barnacle Chamaesipho columna and an encusting brown alga Pseudolithoderma sp. Growth of Pseudolithoderma was enhanced in the presence of live C. columna, which released ammonium at a greater rate than the maximum rate of ammonium uptake by Pseudolithoderma. Algal tissue on barnacle tests had a lower C:N ratio than tissue located more than 2 cm from the nearest barnacle, suggesting the barnacle is an important source of nitrogen for the alga. The role of nutrient exchange in determining ecological patterns of species in marine communities is discussed.