Production and localization of restrictocin in Aspergillus restrictus.

The production and secretion of restrictocin (a cytotoxin that cleaves ribosomal RNA) by cultures of the fungus Aspergillus restrictus was investigated. Previous studies have indicated that restrictocin production in liquid culture coincides with the appearance of differentiated cell structures. A study of the correlation between the appearance of differentiated structures and restrictocin production was conducted with A. restrictus grown on agar medium. Restrictocin was found to be associated with the cell mass of the agar-grown culture (in contrast to liquid cultures), and was first observed when aerial hyphae emerged. Restrictocin levels increased until the time of conidiation, after which they fell off sharply. No restrictocin could be found in the agar medium. The presence of restrictocin upon and within various cell structures was determined by immunofluorescent laser microscopy. This study showed that restrictocin became localized to the conidiophores and phialides during the process of conidiation. Prior to this, restrictocin was found within the hyphae in localized concentrations that may correspond to secretory vesicles.     

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.     

Regulation of the production of polygalacturonase by Aspergillus niger

Synthesis of ethylene in static cultures as well as the effect of endogenous and exogenous ethylene on the synthesis of polygalacturonase byAspergillus niger were determined. This strain produced maximum ethylene amounts when cultured at 30 °C for 3 d. The effect of adding ethylene precursors (citrate-cycle intermediates) on ethylene production was investigated. Best intracellular and extracellular polygalacturonase production was obtained with 2-oxoglutaric, pyruvic and fumaric acids, and with glutamic acid too. Addition of ethylene to the culture medium also increased the synthesis of polygalacturonase, although to a lower degree than when glutamic acid was added.     

Regulation of the production of polygalacturonase by Aspergillus terreus

The synthesis of polygalacturonase (PG) (EC 3.2.1.15) by a strain of Aspergillus terreus was induced by polygalacturonic acid and repressed by glucose, galactose or fructose even in the presence of the inducer. The production of PG increased when the mycelium was washed free of glucose and incubated in a glucose-free medium containing the inducer, a fact that indicated the reversibility of the repression mechanism. When Actinomycin D and cycloheximide were added to the culture medium, the synthesis of PG ceased. PG synthesis increased 43% with the addition of methionine and 64% both with leucine and with tyrosine. Specific productivity with leucine was 210% higher than that of the control as against 149% with methionine and 70% with tyrosine. The results obtained suggest that PG synthesis is regulated by leucine.     

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.     

Naphthalenone production in Aspergillus parvulus.

The naphthalenones asparvenone and its 6-O-methyl ether are produced by Aspergillus parvulus during vegetative growth in batch-mode, shaken cultures in a glucose-glycine-salts medium. The secondary metabolites first appear around the time when hyphae of developing spores begin to make contact with each other, i.e. at a very early stage of culture development. Resuspension in new medium of mycelium that is producing the naphthalenones fails to inhibit production. When A. parvulus is grown in parallel on a glucose-glycine-salts medium and on a glucose-malt-peptone medium, the naphthalenones appear first in the richer medium despite the fact that biomass proliferation is relatively unrestricted in that medium. Radiolabelled [1-14C]-acetate is taken up quickly by the fungus (k = 0.09 min-1) and is incorporated effectively into the naphthalenones.     

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.     

Ochratoxin production by Aspergillus species.

Ochratoxin production was tested in 172 strains representing species in sections Fumigati, Circumdati, Candidi, and Wentii of the genus Aspergillus by an immunochemical method using a monoclonal antibody preparation against ochratoxin A. Ochratoxin A was detected in Aspergillus ochraceus, A. alliaceus, A. sclerotiorum, A. sulphureus, A. albertensis, A. auricomus, and A. wentii strains. This is the first report of production of ochratoxins in the latter three species. Ochratoxin production by these species was confirmed by high-performance thin-layer chromatography and by high-performance liquid chromatography. The chemical methods also indicated the production of ochratoxin B by all of the Aspergillus strains mentioned above.     

Variation in polygalacturonase production among Aspergillus flavus isolates.

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.     

Regulation of protease production in Clostridium sporogenes.

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

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.     

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.