Regulation of restrictocin production in Aspergillus restrictus.

The production of restrictocin (a cytotoxin that specifically cleaves ribosomal RNA) by cultures of Aspergillus restrictus grown in liquid medium was investigated. The function of restrictocin, the method of its accumulation and the mode of resistance to restrictocin in A. restrictus are unknown. Previous studies have indicated that restrictocin accumulates in the medium with culture age. These observations have been extended in this study by cloning the cDNA of the res gene and using this cDNA clone to probe the onset of messenger RNA synthesis in the cells. The results of the Northern analysis were compared to the production and accumulation of restrictocin and morphological differentiation of the cells in culture. Restrictocin was found in the medium at the same time that mRNA was detected in the cells. This suggests that the leader sequence encoded by the cDNA provides an efficient secretion system for the protein. Both the protein and the mRNA were detected coincident with the formation of differentiated cell structures. These structures develop into conidiophores with one layer of sterigmata and conidia forming from the sterigmata. These results suggest that restrictocin is either involved in the process of conidiation or is coordinately regulated with differentiation leading to conidiation.     

Isolation and nucleotide sequence of the Aspergillus restrictus gene coding for the ribonucleolytic toxin restrictocin and its expression in Aspergillus nidulans: the leader sequence protects producing strains from suicide

We describe the cloning and characterization of the gene coding for the ribotoxin restrictocin, from Aspergillus restrictus (gene res, EMBL accession Number X56176). This toxin is a potent inhibitor of protein synthesis in eucaryotes and is of potential interest as a component of immunotoxins. To analyze the mechanism of self-protection in the producing organism, the res gene was cloned into the vector pFB39 and introduced into Aspergillus nidulans. The secretion of active restrictocin from transformants suggests that the pro-toxin is not an active nuclease but is activated during the process of secretion.     

Crystallization and preliminary characterization of mitogillin, a ribosomal ribonuclease from Aspergillus restrictus.

Mitogillin is a ribonuclease secreted by the fungus Aspergillus restrictus. The substrate for mitogillin is a short, universally conserved, sequence in ribosomal RNA. Cleavage of this sequence inactivates protein synthesis. Mitogillin was crystallized by a two-chamber vapor/liquid diffusion method using ethanol as the precipitant. This method has wider potential in the use of volatile organic solvents as precipitants. Crystals of mitogillin diffract X-rays to lattice d-spacings of at least 1.6 A, and belong to the monoclinic space group P2(1), with a = 50.4 A, b = 82.4 A, c = 38.2 A and beta = 99.8 degrees.     

Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.     

Construction and pathogenicity of Aspergillus fumigatus mutants that do not produce the ribotoxin restrictocin

Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA), produces a potent cytotoxjn called restrictocin. To investigate the role of restrictocin in (PA, we have constructed fungal strains in which the res gene has been inactivated by gene disruption. These disruptants lack the specific extracellular ribonucleolytic activity associated with restrictocin, as measured by an in vitro rabbit reticulocyte lysate assay. Western blot analysis of one drsruptant, using an anti-restrictocin monoclonal antibody, confirmed that the toxin is not produced. The growth characteristics of the disruptants could not be distinguished from those of their parental isolates on a variety of culture media. The pathogenicity of two disruptants was assessed in a murine model of IPA. There were no significant differences in mortality when these strains were compared with the parental isolates and an ectopic transformant. In addition, histological examination of infectediung tissue did not reveal any obvious differences in the number or size of fungal colonies or in the polymorphonuclearleucocyte response. Our results demonstrate that restrictocin is not an important virulence factor in this model of IPA.     

Cytochemical localization and ultrastructure of Aspergillus flavus in cottonseed

Cottonseeds having fluorescent fibers were harvested from fields in Arizona and examined utilizing light microscopy and transmission electron microscopy. The occurrence of fluorescent fibers indicated that seeds had been infected by Aspergillus flavus during development. Presence of A. flavus was verified by plating portions of seeds with fluorescent fibers. Hyphae, conidial heads, and conidia were identified readily in differentially-stained cotyledon tissue processed for light microscopy. Utilization of transmission electron microscopy permitted observations on lignified seed coats and cotyledons of mature cottonseeds. Hyphae were located throughout the cotyledon and in the nonlignified layers of the seed coat. The identification of hyphae in cross sections of vessel elements within the seed coat provided ultrastructural evidence supporting the hypothesis that A. flavus may enter seeds via the vascular tissue. Controls for the microscopy studies included observations on cottonseeds with no visual signs of infection and on laboratory-grown cultures of A. flavus. These observations demonstrated that the hyphae localized within fluorescent seeds had features characteristic of A. flavus and that fungal-like structures do not occur within uninfected seeds.     

The observation of plcA mutation and localization in Aspergillus nidulans

To know the function of the plcA gene, which encodes a putative phosphoinositide-specific phospholipase C, in a model filamentous fungus Aspergillus nidulans, it was disrupted thorough homologous recombination and examined. The germination rate of ΔplcA was reduced by approximately 65% and germination of ΔplcA at a lower temperature (25°C) was much slower than germination under normal conditions (37°C), suggesting the plcA is responsible for cold-sensitivity. The hyphal growth of ΔplcA was slightly reduced at 37°C and conspicuously reduced at 25°C. While germinating ΔplcA formed giant swollen spores, and generated short and thick hyphae. The results of the nuclear examination of ΔplcA showed nuclear division with missegregation, and the rate of nuclear division was lower than that of wild type at both 25°C and 37°C. The results of this study showed that plcA is localized to the nucleus through intracellular calcium signaling in A. nidulans. The abnormal nuclear division, resulting from plcA gene deletion, affects conidiation in asexual development. Taken together, these results suggested that plcA is required for normal vegetative growth, morphogenesis, conidiation, and nuclear division in A. nidulans.     

Production of cyclopiazonic acid by Aspergillus flavus Link.

Production of cyclopiazonic acid by Aspergillus flavus is reported for the first time. A procedure for its production by agitated solid substrate fermentation on red wheat is described along with the isolation procedure and physical and chemical properties of this indole derivative. The compound has been found to exert antibacterial activity.     

Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis.

Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single-chain, basic protein of 149 amino acids. The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-terminus in its activity, we have systematically deleted amino-acid residues from both the termini. Three N-terminal deletions removing 8, 15 and 30 amino acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids were constructed. The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized. Removal of eight N-terminal or four C-terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin. The study demonstrates that intact N- and C-termini are required for the optimum functional activity of restrictocin.     

Production of cyclopiazonic acid by Aspergillus tamarii Kita.

Production of the mycotoxin cyclopiazonic acid by Aspergillus tamarii Kita is reported for the first time. Examination of 23 isolates of the fungus showed that 22 produced the toxin under the culture conditions utilized.     

Bioconversion of steroids in mutants of Nocardia restrictus]

Nocardia restrictus grows quickly on synthetic media containing different carbonated substrates (steroids, organic acids). The control of growth parameters of this microbial species allowed the development of the production and selection methodology for mutants unable to grow on androst-4ène-3,17-dione. A stable mutant convert androst-4-ène-3,17-dione in perhydroindan propionic acid without addition of any degradation inhibitor in the medium.     

Effects of amino-terminal extensions and specific mutations on the activity of restrictocin.

The cytotoxic activities of restrictocin with aminoterminal extensions and specific mutations were investigated using in vivo and in vitro systems. Genes were constructed from the cDNA clone of restrictocin which encode: the native form of restrictocin (including the leader sequence); Met-prorestrictocin, in which a codon for methionine was placed before a putative pro region; Met-mature restrictocin, with a methionine codon prior to the mature form of restrictocin; and three mutated forms of Met-mature restrictocin, E95G, E115G/H136L, and H136L. These constructions were placed under the control of the GAL1 promoter and were transformed into Saccharomyces cerevisiae. Transformants were killed, and a new RNA band formed when any of these genes except those containing the H136L mutation were expressed. Restrictocin protein was detected by immunoblot only in cells expressing the native form of restrictocin and the forms containing the H136L mutation. Native restrictocin, Met-prorestrictocin, and Met-mature restrictocin mRNA were translated in an in vitro system resulting in proteins of the expected molecular weight and inactivation of the translation system. Restrictocin was not inactivated by the presence of the leader sequence and the putative prosequence. Amino acid His136 is putatively in the active site of restrictocin by analogy to ribonuclease U2 and the elimination of toxic effects in the S. cerevisiae expression and in vitro translation systems.     

The primary structure of the cytotoxin restrictocin

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS-skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named alpha-sarcin.     

Production of l-DOPA by Aspergillus terreus

The sinefungin producing, pock forming strain Streptomyces incarnatus was shown to be thiostrepton resistant. However, it does not produce thiostrepton and structurally related antibiotics. In this strain, five low copy plasmids of variable sizes were detected with electron microscopy. The strain S. lividans TK24 became thiostrepton resistant upon transformation by one of the plasmids of S. incarnatus.     

Aspergillus niger G proteins: subcellular localization

Aspergillus niger postmitochondrial fraction, which contains high GTPase activity and high GTP binding capacity, has been subjected to subcellular fractionation on a sucrose gradient. A cytosolic and four membranous populations have been separated according to their relative density. The main difficulty has been the characterization of the plasma membrane of the fungus. This fraction, which does not contain any typical enzyme, has been identified after iodination of the outer proteins of protoplasts from A. niger. The immunological detection has shown the occurrence of cytosolic G proteins and membranous small G proteins located not only in the plasma membrane but also in the membranes of the endoplasmic reticulum.     

Production of Kluyveromyces spp. and environmental tolerance induction against Aspergillus flavus

The viability and biomass production of three isolates of Kluyveromyces spp. in six different growth media were studied. All yeast isolates showed good growth in all of the media tested, but nutrient yeast dextrose broth (NYDB 75 %) and molasses soy meal (MSB) media were selected for further analyses. The adaptive response of the yeasts to heat shock and water stress was studied, revealing that 60 min of incubation at 45 °C and a water activity value of 0.95 a_w were the appropriate conditions to adapt these yeasts for subsequent analyses. The physiological adaptation did not affect the ecological similarity between biocontrol agents and pathogen. The adapted yeasts also had a negative influence on the growth of Aspergillus flavus RCM89 mycelia and the accumulation of aflatoxin B₁ levels in vitro. These results have important implications for optimizing the formulation process of proven biocontrol agents against A. flavus. In addition, the applications of physiological methods are necessary for increasing the performance of biocontrol agents. Moreover, the physiological methods could enhance survival under environmental stress conditions of biological control agents.     

Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger

Summary The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both enzymes were located in microbodies. In addition, when the organism was grown on glucose with methylamine as a nitrogen source, amine oxidase activity was detected in the microbodies. These organelles can therefore be designated as peroxisomes.     

Functional characterization and localization of the Aspergillus nidulans formin SEPA

Formins are a family of multidomain scaffold proteins involved in actin-dependent morphogenetic events. In Aspergillus nidulans, the formin SEPA participates in two actin-mediated processes, septum formation and polarized growth. In this study, we use a new null mutant to demonstrate that SEPA is required for the formation of actin rings at septation sites. In addition, we find that a functional SEPA::GFP fusion protein localizes simultaneously to septation sites and hyphal tips, and that SEPA colocalizes with actin at each site. Using live imaging, we show that SEPA localization at septation sites and hyphal tips is dynamic. Notably, at septation sites, SEPA forms a ring that constricts as the septum is deposited. Moreover, we demonstrate that actin filaments are required to maintain the proper localization pattern of SEPA, and that the amino-terminal half of SEPA is sufficient for localization at septation sites and hyphal tips. In contrast, only localization at septation sites is affected by loss of the sepH gene product. We propose that specific morphological cues activate common molecular pathways to direct SEPA localization to the appropriate morphogenetic site.     

Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans.

The PrnB protein is a highly specific proline transporter that belongs to an amino acid transporter family conserved in both prokaryotes and eukaryotes. In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions. Several prnB-gfp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant. Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, four, or eight amino acids, with low potential to form secondary structure elements, were shown to be functional in vivo. A two-linker fusion results in partial complementation at both 25 and 37 degrees C. A four-linker fusion affords almost full complementation at 25 degrees C but partial complementation at 37 degrees C, whereas the eight-linker fusion results in partial complementation at both temperatures but in no GFP fluorescence. These results show that the number of linker amino acids is critical for the correct expression and/or translocation of PrnB-GFP fused proteins to the plasma membrane and for the fluorescence of the GFP. The expression of the four-linker PrnB-GFP transporter was detected and analyzed in vivo by both conventional fluorescence and confocal laser microscopy. This chimeric protein is localized in the plasma membrane, secondarily in large vacuoles found in the swollen conidial end of the germlings, and in other small intracellular structures observed as fluorescent granules. A strong correlation between known patterns of PrnB expression and intensity of PrnB-GFP fluorescence was observed. This work also demonstrates that the GFP fusion technology is a unique tool with which to study the expression and cellular localization of low-abundance transmembrane transporters expressed from their native promoters.