Effect of Different Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture withPseudomonas mendocina

The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD₅₀/mL) and in cocultures with P. mendocina (5000 LD₅₀/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin.     

In vitro Production of Halo Blight Inducing Toxin by Pseudomonas phaseolicola (Burkh.) Dowson

Three pathogenic strains of Pseudomonas phaseolicola (strain 1 and 3 virulent and strain 5 weakly virulent) were tested for their toxic activity. All three strains produced detectable amounts of toxin in vitro. Cultural conditions and length of incubation greatly influenced toxin production. Maximum amount of toxin was produced at 20°C and pH 6.5. Glycerol served as the best carbon source and 1-cysteine as the best amino acid for toxin production.     

Production of a novel and cold-active killer toxin by Mrakia frigida 2E00797 isolated from sea sediment in Antarctica

The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediment in Antarctica was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the psychrotolerant yeast was grown in the medium with pH 4.5 and 3.0% (wt/vol) NaCl and at 15°C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The crude killer toxin activity against the pathogenic yeast M. bicuspidata WCY was the highest when it grew at 15°C in the assay medium with 3.0% (wt/vol) NaCl and pH 4.5. At temperatures higher than 25°C, the killing activity produced by M. frigida 2E00797 was completely lost and after the crude killer toxin was pre-incubated at temperatures higher than 40°C for 4 h, the killing activity was also completely lost. The killer toxin produced by M. frigida 2E00797 could kill only M. bicuspidata WCY, Candida tropicalis and Candida albicans among all the fungal species and bacterial species tested in this study.     

Characterization of a toxin produced by a rhizobacterialPseudomonas sp. that inhibits wheat growth

A toxin produced by a deleterious rhizobacterial pseudomonad that inhibits both winter wheat (Triticum aestivum L.) root andEscherichia coli growth was characterized. The toxin was rapidly deactivated at pH 2 and 12 and by autoclaving (121°C, 15 minutes). Less toxin was destroyed as the temperature and time of exposure decreased, and at 40°C it was stable for at least 24 hours. The toxin was extremely polar and could not be extracted from culture filtrates with organic solvents. The compound eluted after the void volume from a Sephadex G-10 column indicating a molecular weight of less than 700. The toxin adsorbed to Dowex 50W strong cation exchange resin and eluted with 2M NH₄OH. Numerous thin layer chromatography solvent systems were unsuccessful at purifying the toxin. The partially purified toxin inhibited several different microorganisms while the producing strains were resistant. The toxin appears unique to toxins produced by recognized plant pathogenic bacteria.Contribution from the Agric. Res. Serv., U.S. Dept. of Agriculture in cooperation with the College of Agric. and Home Econ., Res. Ctr., Washington State University, Pullman, WA 99164, USA     

Slowly utilized carbon sources enhance botulinic specific toxicity by co-culture of Clostridium argentinense with the non-pathogen Pseudomonas mendocina

The growth and botulinic toxin production of Clostridium argentinense G 89 HT in co-culture with Pseudomonas mendocina were studied using two culture systems with carbon sources of rapid and slow utilization. Growth with glucose in homogeneous co-culture presented a dual-phase progression, with the toxin produced in the slow-growing phase. The extended 50 h growth period of the second phase at low specific growth rate was attributed to the combined metabolization of glucose and a complex carbon source of the alginate type produced by P. mendocina under strongly reducing conditions. With dextrin, the co-culture grew at the lower specific growth rate (μ = 0.03 h⁻¹) for a period lasting 80 h. This fully enhanced the production of toxin with a specific toxicity 2.5 times higher than with glucose in a homogeneous system and 10.7 higher than that of C. argentinense G 89 HT single culture. The heterogeneous co-culture obtained with a dialysis membrane physically separating both bacteria (thereby eliminating the metabolic interaction) produced the lowest levels of growth and toxin of all the cases analyzed.     

Toxin production by a rhizobacterialPseudomonas sp. that inhibits wheat root growth

We studied the production of a toxin inhibitory to both winter wheat (Triticum aestivum L.) root growth andEscherichia coli that was produced by a rhizobacterial pseudomonad. Of several carbon sources tested, the most rapid growth and highest toxin concentrations were obtained with glucose, glycerol, or trehalose. Toxin production was repressed with L-cysteine as the nitrogen source. Toxin was produced during the late exponential and early stationary phase of growth by the bacterium and, contrary to studies with other toxins, was unaffected by Fe and P concentrations in the growth medium. Toxin production by the bacterium was the same at growth temperatures of 25 and 15°C while it produced less at 5°C. If the bacterium was able to grow, it produced toxin. No compound tested induced an increase in toxin production indicating toxin production is constitutive.Contribution from the Agric. Res. Serv., U.S. Dept. of Agriculture in cooperation with the College of Agric. and Home Econ., Res. Ctr., Washington State University, Pullman, Washington 99164.     

Effects of Pichia kluyveri killer toxin on sensitive cells

The killer toxin produced by Pichia kluyveri 1002 kills yeast strains of the genera Candida, Saccharomyces and Torulopsis, including several S. cerevisiae killer strains.Binding of a lethal amount of the toxin to cells of S. cerevisiae SCF 1717 occurs rapidly after toxin addition. After treatment with the toxin for 10 min sensitive cells partially recovered when incubated under conditions that favor protein synthesis. Only after a lag time of 50–90 min sensitive cells changed physiologically. Killing of sensitive cells was characterized by leakage of potassium and adenosine 5-triphosphate, decrease of intracellular pH, and inhibition of the active uptake of amino acids. These effects coincided with cell shrinkage and varied with incubation conditions.Uptake of the amino acid leucine in sensitive cells involved two apparently distinct transport systems (K_m1=0.04mm; K_m2=0.46mm). The toxin showed different effects on these transport systems.     

Alkaline Serine Protease Is an Exotoxin of Vibrio alginolyticus in Kuruma Prawn,Penaeus japonicus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose High Performance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF), and showed maximal activity at pH 10, having a molecular weight of about 33 kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl₂ but partially inhibited by CaCl₂, CuCl₂, CoCl₂, MnCl₂, and ZnCl₂, and not inhibited by ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecyl sulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD₅₀ values of 0.30 and 0.27 μg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy. Received: 12 April 1996 / Accepted: 31 July 1996     

Factors affecting the susceptibility of sensitive yeast cells to killer toxin K1

Optimum conditions for action of the killer toxin K1 on sensitive strainS. cerevisiae S6 were established. Maximum killing was reached in a very narrow pH range of 4.5–4.6. Maximum susceptibility to toxin was displayed by highly energized fresh cells from the early exponential phase in the presence of an external energy source (at least 200 mmol/L glucose). Further, maintenance of maximum membrane potential was necessary for killer action, as documented by decreasing toxin activity in the presence of increasing concentrations of KCl. The killing was strongly stimulated in the presence of millimolar concentrations of Ca²⁺ and Mg²⁺.     

Aflatoxin and Ochratoxin Production by <Emphasis Type="Italic">Aspergillus</Emphasis> Species Under Ex Vivo Conditions

Aspergillus species are increasingly important human pathogens. It is not known whether toxic metabolites of many of these pathogenic species can act as virulence factors in aspergillosis. We examined isolates of aflatoxin and ochratoxin-producing species for toxin production in ex vivo conditions. Seven of the 21 aflatoxin-producing isolates screened produced aflatoxin at 35 and 37°C on the general medium yeast extract sucrose agar (YES). However, none of them produced toxin at these temperatures on brain heart infusion agar (BHA), a medium that mimics human tissue, or on BHA with modified pH or sugar levels. Six of the 12 ochratoxin-producing isolates examined produced toxin at 35°C on YES. All three isolates of A. alliaceus produced ochratoxin on BHA or modified BHA at 37°C. One strain of A. pseudoelegans produced a minute amount of ochratoxin on modified BHA at 37°C. These data indicate that aflatoxin is an unlikely virulence, factor but that ochratoxin may be a potential virulence factor in aspergillosis.     

1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin I

Sticholysin I is an actinoporin, a pore forming toxin, of 176 aminoacids produced by the sea anemone Stichodactyla heliantus. Isotopically labelled ¹³C/¹⁵N recombinant protein was produced in E. coli. Here we report the complete NMR ¹⁵N, ¹³C and ¹H chemical shifts assignments of Stn I at pH 4.0 and 25°C (BMRB No. 15927).     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: PHOSPHOLIPIDS AND PROTEOLIPID

A number of phospholipids known to be constituents of nerve endings were tested for their ability to inactivate botulinum toxin. Substances tested included phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine, phosphatidalethanolamine, β-acyl lysolecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, phosphatidylinositol and cardiolipin. Proteolipid from bovine white matter was also tested. Neutral phospholipids potentiated the toxicity in vivo of botulinum toxin, but they had no effect on the toxicity in vitro. Some, but not all, acidic phospholipids caused loss of toxicity of botulinum toxin in solutions at low pH both in vivo and in vitro. However, none of these substances when incubated with toxin under physiological conditions of temperature, pH and ionic strength, caused loss of toxin potency. The data suggest that none of these phospholipids is likely to be a toxin receptor.     

Volatile and Nonvolatile Maillard Reaction Products between l-Lysine and d-Glucose

The killer toxin produced by the Pichia farinosa KK1 strain was purified by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and reverse-phase HPLC. The molecular weight of the killer toxin was about 25 kd and its isoelectric point was 6.4. A significant amount of carbohydrate was not detected in the purified killer toxin, suggesting that the toxin is not glycosylated. Its N-terminal amino acid sequence showed no homology with other proteins. The stability and efficacy of the toxin’s killer activity was examined. The toxin completely retained activity at pH 2.5 ~ 4.0 and 5°C, but lost activity at higher temperatures. Killer activity increased with increasing NaCl or KC1 concentration, although NaCl was more effective than KCl.     

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

Summary Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25–35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.The assistance of A. Mazza and G.A.R. Mealing is gratefully acknowledged. The trypsin-activated, HPLC-purified CryIC toxin isolated from B. thuringiensis var. entomocidus crystal was a kind gift from M. Pusztai, Institute for Biological Sciences, NRC, Ottawa.     

Production of peroxidase by Inonotus weirii

Summary The white rot fungus Inonotus weirii produced an extracellular peroxidase which was excreted in association with cell growth and in the absence of an inducer. Production of peroxidase was greatly influenced by the carbon and nitrogen source. The highest activities were obtained on glucose-and xylose-based media containing a combination of ammonium nitrate, yeast extract and distiller's spent grain as nitrogen source. The enzyme produced had a molecular weight of 42000, was stable in the pH range 3–8 at room temperature and had optimal activity at pH 3. The fungus Inonotus weirii could be a potential producer of peroxidase for industrial applications in spite of its rather slow production rate.     

The Influence of Carbon Source on the Products of Dissimilatory Iron Reduction

We have examined the influence of carbon source on both the rate of iron reduction and the mineralogy of the reduction products with Shewanella putrefaciens strain W3-18-1. When pyruvate was the carbon source, the secondary products were spherules composed of siderite. When uridine was used as the carbon source, the products were hexagonal plate-like structures identified as iron carbonate hydroxide hydrate, also known as carbonate green rust, a precursor to fougerite. When lactate was used as the carbon source, products were a mixture of iron carbonate hydroxide and magnetite. In terms of reaction stoichiometry, there were differences in the amount of acetate produced depending on the starting organic carbon source. Incubation with pyruvate produced a relatively large amount of acetate compared to incubation with uridine and lactate. There were also differences in the final pH of the cultures. While the pH for incubations with lactate started at 8.6 and ended between 8.0–8.3, the pH of cultures incubated with uridine was found to be almost a full unit lower at the conclusion of the experiment (～7.4). Solubility diagrams based on the chemistry found in our experiments predict that the production of Fe²⁺ _(aq) should always lead to the formation of magnetite. However, strain W3-18-1 produced different minerals depending on the carbon source utilized as the electron acceptor.     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

Effect of culture filtrates ofXanthomonas campestris pv.pelargonii and extracts of geranium stems inoculated withX. c. pv.pelargonii on geranium callus and seedlings

Experiments were designed to determine whetherXanthomonas campestris pv.pelargonii produces a toxin which induces symptoms of bacterial blight in geranium, and is active at the cellular level. Culture filtrates ofX. c. pv.pelargonii were prepared by ethyl acetate extraction and ultrafiltration of the aqueous fraction. Culture filtrates adjusted to several pH values induced maximum disease ratings on geranium seedlings in the pH range 7–10. Geranium callus growth was significantly reduced by the filtrate in the same pH range. An active fraction could also be isolated from diseased tissue. A thin-layer chromatography-callus bioassay system detected toxin activity in the culture filtrate and in extracts of geranium stems inoculated withX. c. pv.pelargonii. Callus growth inhibition was located at R_f = 0.2–0.3 for both sources of toxin. These results suggest thatX. c. pv.pelargonii produces a toxin which causes disease symptoms, is present in diseased tissues, and inhibits callus growth. This opens the possibility of developing resistance to this pathogen by selecting cells insensitive to the toxin and regenerating plants from these cells.     

Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone,Stoichactis helianthus

Summary When present at nanomolar concentrations on one side of a lipid bilayer membrane,helianthus toxin (a protein of mol wt16,000) increases enormously membrane permeability to ions and nonelectrolytes by forming channels in the membrane. Membranes containing sphingomyelin are especially sensitive to toxin, but sphingomyelin isnot required for toxin action. Conductance is proportional to about the 4th power of toxin concentration. Single channel conductances are approximately 2×10^–10 mho in 0.1m KCl. Toxin-treated membranes are more permeable to K⁺ and Na⁺ than to Cl^– and SO ₄ ⁼ , but the degree of selectivity is pH dependent. Above pH 7 membranes are almost ideally selective for K⁺ with respect to SO ₄ ⁼ , whereas below pH 4 they are poorly selective. The channels show classical molecular sieving for urea, glycerol, glucose, and sucrose — implying a channel radius >5 Å. In symmetrical salt solutions above pH 7, theI–V characteristic of the channel shows significant rectification: below pH 5 there is very little rectfication. Because of the effects of pH on ion selectivity and channel conductance, and also because of the rectification in symmetrical salt solutions and the effect of pH on this, we conclude that there are titratable negative charge groups in the channel modulating ion permeability and selectivity. Since pH changes on the side containing the toxin are effective whereas pH changes on the opposite side are not, we place these negative charges near the mouth of the channel facing the solution to which toxin was added.