Environmental and Nutritional Factors Affecting the Production of Rubratoxin B by Penicillium rubrum Stoll

Rubratoxin B can be produced in a semisynthetic medium by Penicillium rubrum under varying environmental and nutritional conditions. Maximum production (552.0 mg/500 ml) was obtained with P. rubrum NRRL A-11785 grown in stationary cultures of Mosseray's simplified Raulin solution supplemented with 2.5% malt extract broth at ambient temperature. Zinc is required at levels of at least 0.4 mg per liter. In the absence of iron sulfate, there was a 50-fold reduction in rubratoxin B production but not in growth. No toxin was produced by this isolate in 5- or 7-liter fermentors.     

Production of [14C]rubratoxin B

[¹⁴C]rubratoxin B was produced by culturing Penicillium rubrum Stoll for 13 days at 22 °C in medium containing [¹⁴C]glucose. The most efficient incorporation of glucose into rubratoxin occurred when Raulin-Thom medium enriched with 2.5% malt extract was supplemented with 2.5% added glucose. The presence of 1.0 mCi of radioactivity in 50 ml of medium with 2.5% added glucose resulted in the production of 38 mg of labeled, chromatographically pure rubratoxin with a specific activity of 0.47 Ci/mole.Rubratoxin B is a hepatotoxic mycotoxin produced by Penicillium rubrum Stoll. It was first isolated by Wilson and Wilson in 1962 (14, 15), and its structure was proposed by Moss in 1968 (11). A simplified procedure for obtaining rubratoxin B in good yield directly from liquid culture media has been described by Hayes and Wilson (6), and there have been numerous investigations of the biological effects of purified rubratoxin B (among others, 4, 7, 9, 12). However due to the insensitivity of the analytical technique for rubratoxin (5), little work has been done to determine the molecular basis of its activity. Determining metabolic products, identifying activated or inactivated toxin complexes or locating binding sites in tissues require a labeled toxin molecule. The purpose of this study was to produce radiolabeled rubratoxin for use in investigations of its various biological activities and also for use in techniques such as autoradiography of tissue sections and radioimmunoassay.Presented in partial fulfillment of the requirements for the degree Master of Science at Iowa State University, Ames, IA 50010.     

Growth and synthesis of rubratoxin by Penicillium rubrum in a chemically defined medium fortified with organic acids and intermediates of the tricarboxylic acid cycle

A sterile glucose-mineral salts broth was fortified with equimolar concentrations (10-³ M) of various organic acids and intermediates in the tricarboxylic acid cycle. Appropriate media were neutralized with 2 N NaOH, inoculated with spore suspensions or mycelial pellets ofPenicillium rubrum and incubated quiescently for 14 days or with shaking for 5 days. Rubratoxins were recovered from culture filtrates by ether extraction and resolved by thin-layer chromatography. Toxin formation in quiescent cultures was enhanced by malonate but was not markedly affected by ethyl malonate, shikimate, and acetate or by isocitrate or oxaloacetate added in the presence of malonate. Citrate, cis-aconitate, -ketoglutarate, succinate, fumarate, and malonate when present in the medium alone or in conjunction with malonate caused a 15 to 50% reduction in rubratoxin formation. Acetyl-CoA (10-⁵ M/flask) caused an 80% increase in toxin yield. Rubratoxin formation in shake cultures was not affected by succinate and malonate. All other combinations of intermediates and malonate caused a 10 to 50% reduction in toxin formation. At 10^–3 M, citrate enhanced rubratoxin B formation and stimulated rubratoxin A production by as much as 100%. Above 10^–3 M, citrate inhibited toxin production. Incorporation of [2-¹⁴C]acetate into rubratoxin was enhanced by malonate, fumarate, and malonate. A combination of pyruvate and malonate produced a 40% increase in [2-¹⁴C]acetate incorporation into rubratoxin. The highest reduction of labeled acetate incorporation (36%) was caused by succinate or -ketoglutarate combined with malonate.     

Bioproduction of rubratoxin in a glucose-mineral salts broth with nutritional supplements and metabolic inhibitors.

A sterile glucose-salts broth fortified with various metabolic inhibitors and nutritional supplements was inoculated with conidia of Penicillium rubrum P3290, and incubated quiescently at 28 degrees C for 14 days. Potassium sulfite and sodium metabisulfite at all test concentrations caused moderate reduction in rubratoxin formation; at high concentrations (greater than or equal to 2.7 X 10(-2)M) accumulation of fungal tissue was also retarded. Production of rubratoxin and cell mass was inhibited by p-aminobenzoic acid; syntheses of toxin were completely blocked by 7.5 X 10(-2)M of the vitamin. Effects of sodium fluoride on P. rubrum cultures grown on inorganic nitrogen sources varied from inhibition of mold growth and (or) rubratoxin A production to reduction in formation of rubratoxin B. With organic nitrogen sources, fluoride caused a 30 and 60% reduction in synthesis of rubratoxins A and B, respectively. Sodium acetate at all test concentrations enhanced formation of rubratoxin; mold growth was enhanced when acetate concentration was larger than or equal to 6.0 X 10(-2)M. A moderate reduction in mold growth was caused by lower acetate concentrations (1.2 X 10(-2)M or 2.4 X 10(-2)M). Sodium arsenite and iodoacetate at test concentrations blocked mold growth and toxin formation; sodium azide and 2,4-dinitrophenol caused a marked reduction in mold growth but inhibited toxin formation completely. However, sodium azide permitted slight growth and toxin formation when mold cultures were incubated for 28 days.     

Rubratoxin production by penicillium rubrum when grown in a synthetic medium containing different sources of carbon and nitrogen

A sterile mineral salts broth was fortified with different additives, inoculated with conidia ofPenicillium rubrum P-13, and incubated quiescently for 14 days or with shaking for 3 to 5 days. Maximal fungal growth and rubratoxin production occurred when the broth contained 20% sucrose. Broth with 10% glucose, 10% fructose, 5% maltose, or 1% asparagine supported formation of substantial amounts of rubratoxin (52.9–78.5 mg/100 ml). When the broth was fortified with glucose plus lysine, arginine aspartic acid, cystine, ammonium citrate, or ammonium phosphate, moderate amounts (27.5–39.5 mg/100 ml) of rubratoxin and mycelium (0.1–1.5 g/100 ml) were produced. Presence in the broth of 5% galactose or starch resulted in accumulation of small amounts (22.2 and 24.6 mg/100 ml, respectively) of rubratoxin and mold tissue (0.70 and 0.5 g/ 100 ml, respectively). Whereas some toxin was recovered from mineral salts broth fortified with lactose or ribose, toxin was not recovered when the mold grew in broth containing mannitol or fumarate. With the exception of gluconate which supported some growth and toxin formation and ethanol which permitted formation of small amounts of toxin, other carbon sources resulted in little or no fungal growth and no toxin formation. Yields of rubratoxin decreased with an increase in amount of agitation or length of incubation ofP. rubrum cultures. Mold growth increased and toxin formation decreased with an increase in volume of culture.     

Production of anti-rubratoxin antibody and its use in a radioimmunoassay for rubratoxin B

Rubratoxin B was coupled to ovalbumin using l-ethyl-3-(3-dimethyaminopropyl) carbodiimide HCl (ECDI) in high concentration at pH 8.O. Under these conditions it was possible to couple 13 moles of rubratoxin B per mole of ovalbumin. The conjugate was used for immunization of rabbits, and anti-rubratoxin antibody was produced. A radioimmunoassay for rubratoxin B was developed which could detect 0.1 g 10 g of toxin using 0.21 g of [¹⁴C] rubratoxin (0.47 Ci/mole, 2.0×10⁹ dpm/g) and 0.125 ml of anti-rubratoxin antibody.Rubratoxicosis was first described in 1957 by Burnside et al. (3) after finding that corn infected with Penicillium rubrum Stoll caused death in swine. Wilson and Wilson (20, 21) reported the isolation of the toxic substance from cultures of P. rubrum in 1962, and the structure of rubratoxin B, shown in Fig. 1, was subsequently determined by Moss (12, 13).Although the gross and histopathological lesions induced by rubratoxin B have been described (3, 19, 22), there are no clinical, pathological or biochemical changes which are specific for the diagnosis of rubratoxicosis (15). It is therefore necessary to isolate the toxin to show its presence. This is only possible with feed samples when there is a large quantity available for extraction, because the 0.5 g sensitivity of the current technique (8) is not sufficient to detect residue from a lethal dose of ingested rubratoxin in animal tissue.It was the purpose of this study to investigate the possibility of adapting the more sensitive radioimmunoassay to the detection of rubratoxin B.Presented in partial fulfillment of the requirements for the degree Master of Science at Iowa State University, Ames, IA 50010.     

Staphylococcus aureus Enterotoxin B Release (Excretion) Under Controlled Conditions of Fermentation

Release of Staphylococcus aureus enterotoxin B (SEB) into the culture medium was initiated during the mid-log phase of growth. A medium consisting of 4% N-Z Amine A (Sheffield), 0.2% dextrose, and 1% yeast extract supported maximum production of SEB. Although pH of the medium during cultivation did not significantly affect the growth curve of the organism, the time required for detectable excretion was affected, as was the final yield. Optimal conditions for SEB production were achieved with pH control at 7.0; alkaline control (pH 8.0) produced only minimal amounts of toxin, whereas acid control (pH 6.0) resulted in 50% reduction in yield. Slightly less SEB was produced when there was no extrinsic pH control, and cultures were buffered only by media constituents and by-products of growth. With pH control at 7.0, deletion of 0.2% dextrose from the medium resulted in 40% reduction in the 8-h yield. There was also a delay in production during early stages of fermentation.     

Different sample treatment approaches for the analysis of T-2 and HT-2 toxins from oats-based media

A LC-DAD method is proposed for the determination of the T-2 and HT-2 toxins in cultures of Fusarium langsethiae in oat-based and other in vitro media. Test media consisted of freshly prepared milled oats to which T-2 and HT-2 toxin stock solutions were added. Different mixtures of extraction solvent (acetonitrile:water and methanol:water), extraction times (30′, 60′ or 90′) and drying methods were investigated. Results showed that extraction with methanol:water (80:20, v/v) for 90 min, drying with N₂ and subsequent analysis by LC-DAD was the fastest and most user friendly method for detecting HT-2 and T-2 toxins production by F. langsethiae strains grown on oat-based media at levels of 0.459 and 0.508 mg of toxin/kg of agar, respectively. The proposed method was used to investigate toxin production of 6 F. langsethiae strains from northern Europe and provided clear chromatograms with no interfering peaks in media with and without glycerol as water activity modifier.     

Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

Differentiation of Clostridium botulinum Types A, B, and E by Pyrolysis-Gas-Liquid Chromatography

Vegetative cells and spores of 10 strains of Clostridium botulinum representing types A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY) medium. Five type E strains were also grown in Multipeptone-sucrose-Nutramino acids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquid chromatography (PGLC) analysis, and the resulting pyrograms were examined for variations in elution patterns between spores and vegetative cells of types A, B, and E grown in the TPSY medium and spores and vegetative cells of type E grown in the TPSY medium and spores and vegetative cells of type E grown in TPSY and MSN media. Growth and toxin production of all 10 strains of C. botulinum were investigated by using a modified dialysis sac culture technique. The dialysate supernatant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from the dialysate was also subjected to PGLC analysis. Control samples consisting of (i) noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii) 1.0 mg of partially purified toxin were also analyzed by PGLC. Differences between pyrograms of cultures were suitable for positive identification at the type level but not at the strain level. Pyrograms permitting differentiation were also obtained between spores and vegetative cells as well as between the same cultures grown in different media. The dialysis sac technique was useful in detecting growth but not toxin production of C. botulinum.     

Differential Effects of Lithium Chloride on In Vitro Growth of Clavibacter michiganense subsp. nebraskense Depending upon Inoculum Source

The bacterium Clavibacter michiganense subsp. nebraskense (Corynebacterium michiganense subsp. nebraskense) was grown in broth cultures and inoculated into corn plants. The plating efficiency of cells from broth cultures was essentially the same on nutrient broth-yeast extract and the semiselective medium for this bacterium, CNS. However, when cells were isolated from Goss bacterial wilt- and blight-infected corn, very few were recovered on CNS compared with the amount recovered on nutrient broth-yeast extract agar. When lithium chloride was omitted from the CNS, recoveries from infected corn were nearly the same as on nutrient broth-yeast extract agar. No other ingredient of CNS was inhibitory, nor did substitution of other salts for lithium chloride cause equal inhibition. The amount of inhibition was proportional to lithium chloride concentration. The inhibition by lithium chloride occurred with several strains of the bacterium isolated from one corn cultivar and with one of the strains recovered from three different cultivars of infected corn.     

Induction of Chlorosis,ROS Generation and Cell Death by a Toxin Isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.     

Production of Escherichia coli Heat-labile Enterotoxin in Fermenter Dialysis Culture

Production of Escherichia coli heat-labile enterotoxin was investigated with one porcine and one human strain in three different media under different cultivation conditions. Cultivation in aerated fermenters at pH 7·0 yielded 10–20 times more enterotoxin/ml of culture fluid than cultivation in shake flasks. A trypton-yeast extract medium was optimal in fermenter cultures. Comparatively good yields of enterotoxin in fermenters were also obtained in a glucose-salts medium. Continuous feeding of glucose and salts during fermenter cultivation resulted in a lower production of enterotoxin/mg of bacterial cells. Since this decrease in specific yield could be reversed by using dialysis culture, it was concluded that inhibition of toxin formation was due to the accumulation of extracellular low molecular weight metabolites. The highest yield of enterotoxin in dialysis culture was 80 ED₅₀ ml⁻¹ (rabbit jejunal loop test) which is at least eight times more toxin than in ordinary fermenter culture and 80 times more toxin than in shake flask cultures.     

Effect of environmental stress on Clostridium difficile toxin levels during continuous cultivation.

A method for the continuous culture of Clostridium difficile has been described. It has been shown that subjecting continuous cultures of this microorganism to environmental stress results in increased levels of toxin in culture medium. Factors found to cause this release include alteration of the Eh from --360 to +100 mV or increasing the temperature from 37 to 45 degrees C. The increased toxin levels were not associated with a change in viable cell density or the numbers of spores present. Additional studies have shown that subinhibitory concentrations of vancomycin and penicillin, but not clindamycin, also cause an increase in toxin levels during continuous culture. The increase in supernatant toxin levels occurs concomitant with a decrease in sonicated cell extract toxin levels. The data suggest that a number of factors can cause a release of toxin from C. difficile into the surrounding medium.     

Effect of environmental stress on Clostridium difficile toxin levels during continuous cultivation.

A method for the continuous culture of Clostridium difficile has been described. It has been shown that subjecting continuous cultures of this microorganism to environmental stress results in increased levels of toxin in culture medium. Factors found to cause this release include alteration of the Eh from --360 to +100 mV or increasing the temperature from 37 to 45 degrees C. The increased toxin levels were not associated with a change in viable cell density or the numbers of spores present. Additional studies have shown that subinhibitory concentrations of vancomycin and penicillin, but not clindamycin, also cause an increase in toxin levels during continuous culture. The increase in supernatant toxin levels occurs concomitant with a decrease in sonicated cell extract toxin levels. The data suggest that a number of factors can cause a release of toxin from C. difficile into the surrounding medium.     

Phenolic accumulation and peroxidase activity inin vitro selected alfalfa callus cultures resistant to filtrate ofFusarium spp.

Changes in the phenylalanine ammonia-lyase (PAL) activity, accumulation of phenolic acids and ionically-bound peroxidase activity in thein vitro selected embryogenic and nonembryogenicMedicago sativa callus cultures resistant to the filtrate ofFusarium spp. were found. The PAL activity in bothin vitro selected cultures during a 4-week cultivation on a medium with phytotoxins was higher than in the control calli grown on a medium without toxin. The filtrate fromFusarium spp. evoked an increase in the contents of all determined phenolic acids in the selected calli. They occurred predominantly bound as esters. The most pronounced portions in the elevated acids level were of ester-bound p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids. The ionic cell wall-bound peroxidase activity in both selected calli cultivated on a medium with a filtrate was twice as high as the activity determined in the control cultures. The activity of soluble peroxidase was not influenced by challenge with a filtrate. No significant differences were found between thein vitro selected embryogenic and nonembryogenic alfalfa callus cultures in the response to the phytotoxic filtrate.     

Selection of Citrus limon cell culture variants resistant to the mal secco toxin

Embryogenic cell cultures of Villafranca lemon capable of growth were selected in the presence of toxin produced by the fungus Phoma tracheiphila, the causal agent of Mal secco. Mal secco is a serious tracheomycotic disease of lemon (Citrus limon Burm. f.) and citron (C. medica L.). Rigorous clonal selection with the variant line was conducted for six consecutive subcultures. Viability of cell lines was monitored by the use of fluorescein diacetate. The stability of the resistance was examined after growth on non-selective medium for three subcultures. The variant line Var. 1.117 showed stable resistance. Cells of the resistant variant line maintained their enbryogenic capacity. Callus induced from the resulting somatic embryos also displayed resistance to the toxin.     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Selection of lactose-adapted cells in vinca minor and Dantura innoxia cultures; location and characterization of β-galactosidase and lactase activities

Lactose-adapted cells were obtained from Datura innoxia sucrose growing calli cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous population in D. innoxia cultures.In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown cells as well in Datura lactose-adapted cells cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for thecells; it was released into the culture medium and not metabolized in Vinca cultures while it was metabolized in Datura cultures at the end of the culture period.