Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucroselow salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Effect of zinc on adenine nucleotide pools in relation to aflatoxin biosynthesis in Aspergillus parasiticus.

The adenylic acid systems of Aspergillus parasiticus were studied in zinc-replete and zinc-deficient media. The adenosine 5'-triphosphate levels of the fungus were high during exponential phase and low during stationary phase in zinc-replete cultures. On the other hand, the levels of adenosine 5'-diphosphate and adenosine 5'-monophosphate were low during exponential phase of growth and high during stationary phase. The adenosine 5'-triphosphate levels during exponential phase may indicate higher primary metabolic activity of the fungus. On the other hand, high adenosine 5'-monophosphate levels during stationary phase may inhibit lipid formation and may enhance aflatoxin levels. The inorganic phosphorus content was low in a zinc-replete medium throughout the growth period, thereby favoring aflatoxin biosynthesis. The energy charge during the exponential phase was high but low during the stationary phase. In general the energy charge values were lower because of high adenosine 5'-monophosphate content.     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.     

Aflatoxin formation,lipid synthesis,and glucose metabolism by Aspergillus parasiticus during incubation with and without agitation

Glucose utilization, growth of mold, and synthesis of aflatoxin and total lipid by Aspergillus parasiticus were studied with cultures that were incubated statically and with agitation. With both cultural conditions, maximal toxin formation occurred at 5 days which coincided with the end of rapid mold growth and rapid uptake of glucose. The toxin concentration decreased as incubation continued. The pattern for formation and depletion of total lipid was similar to that for aflatoxin. Maximal yields of toxin and of total lipid did not coincide with maximal production of mold mycelium. Incubation with agitation enhanced mold growth, consumption of glucose, and production of aflatoxin and total lipid during the first 3 days. Generally, more growht occured in agitated cultures, but maximal yields of aflatoxin and total lipid were lower than in quiescent cultures. The need for limited, but not excessive, O₂ for synthesis of aflatoxin and lipid also was demonstrated by varying the volume of medium in flasks that were incubated quiescently. Incorporation of [1-¹⁴C] glucose into aflatoxin indicated that limiting the O₂ supply and thereby favoring glucose catabolism via the Embden-Meyerhof pathway enhanced toxin formation. Aflatoxin formation also was greater when oxidative respiration of the mold was restricted by a metabolic inhibitor. Results suggest that the degree of aeration of the culture is important in controlling biosynthesis of aflatoxin.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Pyridine nucleotides in normal and nicotinamide depleted adrenal tumor cell cultures

Mouse adrenal tumor cell line Y-129 was grown in cell culture in medium without nicotinamide. Inhibition of growth occurred after the second subculture in the vitamin-deficient medium. Pyridine nucleotides were measured in control and nicotinamide-starved cultures. DPN⁺ decreased to less than 10% of the normal level after 7 days of vitamin starvation. TPNH dropped to 25% of its normal level in the same period. Plating efficiency decreased from 20% in controls to 10% in 7-day nicotinamide-starved cultures. Steroid production was reduced by approximately 50% in the starved cultures. Sensitivity of the cultures to the lethal effects of the nicotinamide antagonist, 3-acetylpyridine, was inversely proportional to the pyridine nucleotide content of the cells. Reconstitution of nicotinamide in depleted cultures caused a rapid renewal of the pyridine nucleotide levels and complete protection against the effects of 3-acetylpyridine. Recovery and subsequent growth of the cultures did not restore the decreased capacity to produce steroids. Nicotinic acid was not utilized by the cells for the synthesis of pyridine nucleotides in depleted cultures and azaserine did not inhibit the utilization of nicotinamide indicating that DPN⁺ was synthesized directly via nicotinamide ribonucleotide.     

Coconut as a Medium for the Experimental Production of Aflatoxin

Fresh, grated coconut has been found to be an excellent medium for aflatoxin production by Aspergillus flavus. Under optimal conditions, yields of 8 mg of total aflatoxin per g of substrate were obtained. Continuous agitation of the growth medium under moist conditions at 24 C produced highest yields. Aflatoxin was assayed both biologically and chromatographically. The aflatoxin content of cultures varied biphasically with the duration of incubation. It is suggested that this pattern could result from the sequential operation of factors promoting aflatoxin formation on the one hand and a detoxifying mechanism on the other.     

Induction of aflatoxin biosynthesis inAspergillus parasiticus by ascorbic acid-mediated lipid peroxidation

The effect ofl-ascorbic acid on the biosynthesis of aflatoxin inAspergillus parasiticus was studied. Ascorbic acid at lower concentrations did not inhibit the growth of fungus but markedly induced aflatoxin biosynthesis. At a concentration of 1000 ppm of ascorbic acid, 4.8-fold higher levels of aflatoxin were detected. Copper did not enhance the induction of toxin synthesis by ascorbic acid when added to the growth medium. Ascorbic acid at 1000 ppm was also found to induce aflatoxin synthesis in resting mycelia. Chloroform (1% vol/vol) was found to induce aflatoxin synthesis under similar conditions. Ascorbic acid in the presence of ferrous ion can cause lipid peroxidation, which in turn is responsible for the induction of aflatoxin synthesis. During the induction of aflatoxin synthesis by ascorbic acid, the uptake of carbon source (acetate) was not affected. This observation suggests that on ascorbic acid treatment a precursor or an intermediate of aflatoxin biosynthesis is synthesized in vivo and is responsible for the higher levels of toxin without increasing the uptake of acetate.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Cowpeas as growth substrate do not support the production of aflatoxin byAspergillus sp

A number of 21Aspergillus sp. strains isolated from cowpeas from Benin (Africa) were characterized by RAPD methodology. Seven of these strains grouped withA. flavus in the dendrogram generated with the RAPD data. Only three were able to produce aflatoxin in significant amounts. Twelve other isolates grouped withA. parasiticus. All of these strains except 3 produced aflatoxin. Two additional strains neither fit with theA. flavus group, nor theA. parasiticus group according to their RAPD pattern. Both did not produce aflatoxin in measurable amounts. Generally the aflatoxin positive strains produced high amounts of aflatoxin after growth on YES medium. However after growth on cowpea based medium aflatoxin biosynthesis was strongly ceased, albeit the growth of the colony was only partly reduced. This was true for media made either with the whole cowpea seed or with cowpea seed without seed coat. Interestingly when the cowpea medium was heat sterilized the fungus was able to produce high amounts of aflatoxin. This, however, was not the case after the use of gamma irradiation as sterilization method for the medium. The expression of thenor- 1 gene, which is one of the early genes involved in aflatoxin biosynthesis, was significantly repressed after growth on gamma irradiated cowpea medium in contrast to YES medium. This study was part of the project “Capability Building for Research and Quality Assurance in Traditional Food Processing in West Africa”     

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-β-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C₁ metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.     

Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum

Concentrations of adenine and pyridine nucleotides and the associated charge values were examined in extracts of mycelium of Penicillium notatum during vegetative growth and reproductive development promoted by the addition of Ca²⁺ (10 mmol dm^-3). The significant increase in adenylate energy charge promoted by Ca²⁺ was due to a fall in intracellular AMP and a concomitant rise in ATP concentration. Intracellular concentrations of NADH and NAD fell within 1 h of the addition of Ca²⁺. The catabolic reduction charge was unchanged by Ca²⁺ whilst the anabolic reduction charge increased in Ca²⁺-induced mycelium due to lowered intracellular NADP concentration. Reduced concentration of NADPH in Ca²⁺-induced mycelium, relative to the vegetative controls, lowered the phosphorylated nucleotide fraction. The results are discussed in relation to metabolic economy during morphogenesis in P. notatum.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Pyridine nucleotide synthesis in 3T3 cells.

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.     

Growth and aflatoxin production by Aspergillus parasiticus when in the presence of Streptococcus lactis

Streptococcus lactis was grown with Aspergillus parasiticus in modified APT broth. Three inoculation procedures were used: (a) S. lactis was grown 3 days, then conidia of A. parasiticus were added (SLAP), (b) both organisms were added simultaneously (ST) and (c) A. parasiticus was grown 3 days, then S. lactis was added (APSL). At 3, 6 and 10 days of incubation, contents of flasks were analyzed for growth of each organism, pH of broth and aflatoxin content. S. lactis did not survive past 3 days when grown alone. In ST cultures, S. lactis grew to the same extent as in the control at 3 days; it remained viable at a low level through 10 days. In APSL cultures, S. lactis growth was inhibited at 3 days but the bacterium survived through 7 days (10 days of mold growth) at reduced numbers. At 3 days there were no appreciable differences in growth of A. parasiticus. At 6 days, in ST and SLAP cultures, growth of the mold was inhibited, while in the APSL culture growth increased over that in the control. At 10 days, growth of mold was somewhat increased over the control in all test conditions. The pH of broth in the A. parasiticus control and APSL culture was 6 at 3 days, dropped to 4.5–4.6 at 6 days and rose to 7 by 10 days. In ST and SLAP cultures, the pH was at 4.1 at 3 days and rose to pH 7 by 10 days. Aflatoxin (B₁ plus G₁) content was lowest at 3 days and increased at 6 days. Between 6 and 10 days two patterns were observed. In APSL and SLAP cultures, aflatoxin content decreased, while it increased in the ST culture. These patterns occurred when aflatoxin content was expressed on a total or per gram of dried mycelium basis. At 3 days the amounts of aflatoxin B₁ and G₁ were approximately equal. Between 3–6 days the amount of G₁ increased more rapidly than that of B₁. Between 6 and 10 days in the ST culture, the amount of G₁ increased at a slower rate than that of B₁ while in SLAP and APSL cultures, the amount of G₁ decreased more rapidly than that of B₁. When a different lot of the same medium was used, aflatoxin production was greatly reduced. The pH of broth at all test conditions rose through the incubation period.     

Effect of aflatoxin G1 on germination, growth and metabolic activities of some crop plants.

The effect of different concentrations of aflatoxin G1 on growth and germination of Zea mays and Vicia faba seeds, as well as on some biochemical parameters viz chlorophyll, carotenoid, protein and lipid content of seedlings, were studied. Inhibition of seed germination and seedling growth of maize and broad bean increased with increases in toxin concentration. A reduction in carbohydrates in the shoot systems of maize and broad bean was accompanied by a corresponding reduction in chlorophyll content. The total proteins and total lipids of V. faba were significantly greater at a 10 micrograms/ml concentration of aflatoxin G1, whereas in Z. mays significant inhibition (p < 0.05) was observed. At 5.0 micrograms/ml aflatoxin G1 lipids and proteins were reduced in both plants but the effect was less obvious at lower concentrations.