Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.     

Aflatoxin B1 Induction of Lysogenic Bacteria

A technique for biological verification of aflatoxin B(1) was developed based on toxin-mediated induction of lysis in a lysogenic strain of Bacillus megaterium NNRL B-3695. Reduction of culture turbidity was determined at various concentrations of toxin. Incubation of 1.1 x 10(-4) g (dry weight) of cells/ml of growth medium containing 25 mug of B(1) per ml at 37 C reduced initial turbidity 0.20 absorbance units in 4 hr. If the bacterial lysate of the lysogenic strain, after a 2-hr incubation with 25 mug of B(1) per ml, was plated with a sensitive B. megaterium strain (NRRL B-3694), plaque-forming units increased approximately 150 times relative to the control. Comparable testing of the effects of aflatoxin on the nonlysogenic, sensitive strain demonstrated that 75 mug of B(1) per ml neither induced lysis nor plaque-forming units. Although induction is not an exclusive property of aflatoxin B(1), the differential response of the lysogenic and sensitive Bacillus strains to B(1) offers a unique and rapid technique for biological verification of the toxin.     

Prophage induction and filamentation in Bacillus thuringiensis caused by the genotoxic mycotoxin aflatoxin B1

Cultures of the lysogenic strain of Bacillus thuringiensis var. tolworthi were made in the presence of various drugs. The determination of bacterial size and plaque forming units (by using an indicator strain of B. thuringiensis var. galleriae) as well as colony forming units were then performed. Treatment of lysogenic cells by aflatoxin B1: provokes the formation of elongated cells (filamentation); induces a pathway that leads to the induction of prophage. Results of the present study indicated that filament formation and bacteriophage induction are two commonplace effects that occur in virtually every member of this cellular population exposed to low doses of certain drugs such as aflatoxin B1 (10 micrograms/ml); all of which have in common the ability to produce damaging changes in DNA. The following findings support the hypothesis that error-prone repair mechanisms seem to be present in B. thuringiensis as in Escherichia coli.     

Heat activation of Bacillus spores by the use of microwave irradiation

A study was conducted in which microwave irradiation and conventional waterbath treatment were compared as to their efficiency for heat-activating Bacillus spores. Spore suspensions were prepared from B. brevis, B. cereus, B. licheniformis, a lysogenic strain of B. megaterium (NRRL-B-3695), two strains of B. stearothermophilus, and B.Subtilis. Suspensions were either irradiated for 30 sec in a microwave oven, or conventionally heat-treated in the waterbath for 60 min at 60 degrees C, the serially diluted and plated onto nutrient agar. Colonies of each species from each treatment were isolated, and cultures were inoculated into several biochemical media. Spore suspensions heat-activated by microwave irradiation resulted in plate counts that were from 3% to 24% greater than from suspension heat-activated by conventional mean (60 degrees C for 60 min). There were no observed alterations in biochemical activities in any of the representative colonies from either of the two treatments. No induction of bacteriophage from lysogenic B. megaterium NRRL-B-3695 was observed in colonies from either of the two treatments. Microwave irradiation appears to be more efficient, less time-consuming, and at least as effective as heat activation by conventional waterbath treatment for Bacillus spores.     

Aflatoxin B1 effect on beta-D-galactosidase in some starter cultures.

Six starter cultures were examined for their ability to induce beta-D-Galactosidase activity. The maximum (713 x 10(-4) units/ml) and minimum (140 x 10(-4) units/ml) enzyme activity was shown by Lactobacillus bulgaricus and Streptomyces thermophilus, respectively. The maximum inhibition by aflatoxin B1 of induction and activity of enzyme in vitro was observed at 60 micrograms/ml in all cultures. However, low levels of the toxin stimulated induction of the enzyme in Streptococcus lactis and S. thermophilus.     

Effects of Mitomycin C and Thymidine Deprivation on Lysogenic and Sensitive Strains of Bacillus megaterium

A triple auxotroph of Bacillus megaterium strain KM was lysogenized with a phage suspension from B. megaterium 899a. The lysogenic and phage-sensitive derivatives of KM were found to die at the same exponential rate during thymineless incubation, despite the fact that the lysogenic strain became induced. The lysogenic strain was also induced by mitomycin C, and died at an exponential rate which was approximately twice that of the sensitive strain. With both strains, the lethality of mitomycin C was the same in the presence and absence of thymidine; thymidine was required for maximal phage production. Mitomycin C preferentially inhibited deoxyribonucleic acid (DNA) synthesis of both strains for the first 60 min. The (DNA) synthetic ability of the lysogenic strain was subsequently restored, due to phage production. Since there was no evidence that sensitive strains of KM contained other inducible elements (prophage or probacteriocins), it is concluded that both thymineless death and mitomycin C death can occur via mechanisms not involving induction.     

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro.

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Morphological Alterations in Bacillus megaterium as Produced by Aflatoxin B1

Morphologically abnormal cells were produced by Bacillus megaterium NRRL B-1368 in response to aflatoxin B(1). Filamentous forms were characterized by early granulation and unusually large and numerous deposits of poly-beta-hydroxybutyric acid within the cells. Pantoyl lactone was without effect as a reversing agent for the observed inhibition of cell septum formation. B. megaterium cells and spores produced on toxic (3.8 mug of aflatoxin B(1) per ml) and nontoxic Trypticase Soy Broth and Trypticase Soy Agar (TSA) were observed by using phase contrast and electron microscopy. Transfer of aberrant forms to nontoxic TSA yielded macrocolonies with daughter cells morphologically indistinguishable from untreated cells. Agar slide cultures of filamentous cells transferred to nontoxic TSA indicated that normal cells were formed. Electron photomicrographs showed a decreased number of mesosomes in filamentous cells as compared to control cells. There were no observable morphological differences in spores formed on toxic or nontoxic TSA.     

Aflatoxin B1 Uptake by Flavobacterium aurantiacum and Resulting Toxic Effects

Removal of aflatoxin B(1) from liquid cultures by resting and growing cells of Flavobacterium aurantiacum NRRL B-184 was studied. Spectrophotometic and thin-layer techniques served as aflatoxin assays. Cells grown in the presence of 5 ppm or higher levels of aflatoxin developed aberrant morphological forms. These toxin concentrations partially inhibited growth, and the nature of the inhibition suggested that aflatoxin interfered with cell wall synthesis. Incubation of 1.0 x 10(11) resting cells per milliliter with 7.0 mug/ml of aflatoxin B(1) during a 4-hr period facilitated complete toxin removal from a buffered aqueous medium. Autoclaved cells and cell wall preparations could remove a fraction of the aflatoxin of a test system. However, the toxin removed by autoclaved cells and cell walls could be extracted by washing with water but the aflatoxin B(1) removed by intact cells could not be extracted into the liquid phase. The uptake of aflatoxin B(1) by resting cells was sensitive to temperature and pH. Ruptured preparations of F. aurantiacum were not able to remove or modify the aflatoxin in an aqueous solution.     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Reverszal of grwoth inhibition of Bacillus megaterium dut to aflatoxin by coffee and tea extracts

Growth inhibition of Bacillus megaterium by aflatoxin B₁ is employed in semi-quntitative assay of the toxin,. Aqueous extracts of tea and coffee were found in interfere with this assay, which indicated that tea and coffee contain factors(s) which can reverse the aflatoxin toxicity on B. megaterium     

Survey of the Sensitivity of Microorganisms to Aflatoxin

Among the 329 microorganisms tested for aflatoxin sensitivity were 30 genera of bacteria, 34 genera of fungi, 4 genera of algae, and 1 protozoan. Twelve species of the genus Bacillus, a clostridium, and a streptomycete were inhibited when 30 mug/ml of crude aflatoxin (36% pure) was incorporated into the growth substrate. A strain of Bacillus brevis and two of B. megaterium were most sensitive to aflatoxin, being inhibited at 10 and 15 mug/ml, respectively.     

Increased cytotoxicity of food-borne mycotoxins toward human cell lines in vitro via enhanced cytochrome p450 expression using the MTT bioassay

Eight food-borne mycotoxins epidemiologically implicated in human disease were tested for their cytotoxic effects on human cells previously immortalised and transfected to introduce human cytochrome p450 (CYP 450) genes. Such cells retain many characteristics of normal cell growth and differentiation while simultaneously having the potential of either increasing or decreasing the metabolic activity (cytotoxicity) of the challenging mycotoxins. The MTT assay provided an indication of cytotoxicity. Of the nine CYP450s introduced CYP1A2 was most effective, rendering the cells 540 times more sensitive than the control cells to aflatoxin B1, 28 times more sensitive to aflatoxin G1 and 8-fold more sensitive to ochratoxin A. CYP3A4 resulted in the cells being 211 times more toxic to aflatoxin B1 and 8-fold more toxic to aflatoxin G1 while CYP 2A6, CYP 3A5 and CYP 2E1 also produced observable effects. No increase in metabolic activity was found using cyclopiazonic acid, deoxynivalenol, fumonisin B1, patulin or T-2 toxin. CD5Os were calculated for the mycotoxins against the non-CYP-introduced control cells. There was almost a five order of magnitude difference between the most toxic, T-2 toxin (CD50 0.0057 microgram/ml) and the least toxic, fumonisin B1 (CD50 476.2 micrograms/ml). In vitro biological assays thus provide an excellent system for quantifying the often low CD50s expressed by mycotoxins in foods.     

Uptake of aflatoxin B1 and T-2 toxin by two mycotoxin bioassay microorganisms: Kluyveromyces marxianus and Bacillus megaterium

Uptake of aflatoxin B1 (AFB1) and trichothecene T-2 toxin from growth medium by mycotoxin bioassay strains of Klutyveomyces marxianus and Bacillus megaterium was assessed by incubating, washing, and sonicating the cells, extracting samples with chloroform, and analysing the extracts by a combination of high-performance thin-layer chromatography (HPTLC) and fluorescence densitometry. Using cultures of K. marxianus, the entire AFB1 dose was recovered and no AFB1 metabolites were detected. Less than 1% of the AFB1 was recovered from the cells, and AFB1 did not inhibit growth. Methanol in the incubation medium had no significant effect on the levels of AFB1 associated with K. marxianus cells. The entire dose of T-2 toxin was also recovered from K. marxianus cultures, and no metabolites were detected; again, less than 1% of T-2 toxin was cell-associated, but growth was completely inhibited. AFB1 partially inhibited the growth of B. megaterium; approximately 12% of the dose could not be recovered, and no AFB1-related metabolites were detected. Methanol increased the levels of recoverable AFB1 associated with B. megaterium cells. In the case of T-2 toxin, around 8% of the dose was not recovered, and no metabolites were detected; growth of B. megaterium was stimulated. These results suggest irreversible binding of both toxins, or derivatives of them, to the cells of B. megaterium.     

Bordetella pertussis bacteriophage

For the first time Bordetella pertussis bacteriophage was isolated, and its presence was confirmed by electron microscopy and by agar layer titration. The lysogenic strains were activated by their treatment with mitomycin C in a dose of 4.5 mg/ml. The phage system of the Bordetella genus, heretofore unknown, has been revealed: Bordetella pertussis phage lyzed all the tested strains of Bordetella parapertussis (25 strains) and could be passaged in these strains. The phage formed turbid and transparent negative colonies 0.1 mm and 0.15 mm in size. The phage titer (e. g., in strain No. 3865) was 1 X 10(10). The lysogenic variants of Bordetella pertussis, capable of spontaneous release of the phage, were obtained. These variants were characterized by changes in some of their phenotypical properties, e.g., the increased content of certain toxic substances and increased virulence.     

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.     

Potential Significance of Lysogeny to Bacteriophage Production and Bacterial Mortality in Coastal Waters of the Gulf of Mexico

The potential effect that induction of lysogenic bacteria has on bacteriophage production and bacterial mortality in coastal waters was investigated, and we present estimates for the percentage of lysogenic cells in a natural aquatic bacterial community. Various concentrations of mitomycin C and exposure times to UV C radiation (UV-C) (wavelength of 254 nm) were used to induce the lytic cycle in lysogenic cells of natural communities of marine bacteria. UV-C treatment occasionally resulted in phage production, but phage production induced by UV-C was always less than that caused by the addition of mitomycin C. There was no evidence that high growth rates of bacteria resulted in lysogenic phage production. The burst size of cells induced by mitomycin C was determined by transmission electron microscopy and ranged from 11 to 45. Dividing the induced phage production by the burst size provided an estimate of the number of lysogenic bacterial cells, which ranged from 0.07 to 4.4% (average, 1.5%) of the total bacterial population. The percentages of lysogenic bacteria that were induced by mitomycin C were similar for samples collected nearshore from the pier of the Marine Science Institute (chlorophyll a, 1.6 to 2.9 (mu)g liter(sup-1)) and in relatively oligotrophic water (chlorophyll a, 0.2 to 0.9 (mu)g liter(sup-1)) collected 25 to 100 km offshore. By using a steady-state model, if all lysogenic bacteria were induced simultaneously, 0.14 to 8.8% (average, 3.0%) of the total bacterial mortality would result from induction of lysogenic cells. If mitomycin C induces all or the majority of lysogenized cells, our results imply that lysogenic phage production is generally not an important source of phage production or bacterial mortality in the coastal waters of the western Gulf of Mexico.