Abstract: Aim: To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions.
Methods and Results: In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium.
Conclusions: Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses.
Significance and Impact of the Study: The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.
Abstract: Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings produce a 1197 Da toxin, named amylosin. Nuclear magnetic resonance (NMR) data showed that amylosin contains a chromophoric polyene structure and the amino acids leucine/isoleucine, proline, aspartic acid/asparagine, glutamic acid/glutamine and tyrosine. A quantitation method for amylosin was developed using commercially available amphotericin B as a reference compound and a known concentration of amylosin determined by NMR with the electronic reference to access in vivo concentration (ERETIC) method. Purified amylosin inhibited motility of boar sperm cells at an exposure concentration of 135 nM and hyperpolarized their cell membrane and depolarized their mitochondria at exposure to concentration of 33-67 nM for 10 min. In a 3-d exposure time only 27 nM of amylosin was needed to provoke the same toxicity functions. Amylosin was cytotoxic to feline lung cells at concentrations of <170 nM. Purified amylosin provoked adenosine 5'-triphosphate (ATP)-independent cation influx into isolated rat liver mitochondria (RLM), inducing swelling of the mitochondria at concentrations of 200 nM K(+) or >250 nM Na(+) medium. In the K(+)- or Na(+)-containing medium, amylosin uncoupled RLM, causing oxidation of pyridine nucleotides (PN), loss of the mitochondrial membrane potential, and suppressed ATP synthesis. Purified amylosin produced cation channels in black-lipid membranes (BLMs) with a selectivity K(+)>Na(+) at a concentration of 26 nM, i.e. the same concentration at which amylosin was toxic to boar sperm cells. The amylosin cation channels were cholesterol- and ATP-independent and more effective with K(+) than with Na(+). We propose that the toxicity of amylosin may be due its ionophoric properties, representing the first K(+)/Na(+) channel-forming substance reported from B. amyloliquefaciens.
Abstract: Valinomycin and cereulide are bacterial toxins with closely similar chemical structure and properties but different toxic effects. Emetic poisoning is induced by cereulide but not by valinomycin. Both are specific potassium ionophores. Such compounds may affect mitochondrial functions. Both compounds cause a potassium-dependent drop in the transmembrane inner membrane potential due to the uptake of K+ as positively charged ionophore complex. Valinomycin is more potent than cereulide at high [K+] (>80 mM), whereas cereulide in contrast to valinomycin is active already at <1 mM. With cereulide, there is a substantial lag, while valinomycin acts without lag. Both ionophores induce mitochondrial swelling in the presence of K+, in the case of cereulide with a lag. These toxins strongly inhibited respiration at the level of complex IV when used at higher concentrations than that used for detection of ionophoretic transport of K+. At high [KCl] (120 mM), valinomycin was more potent than cereulide both as ionophore and inhibitor, but at low [KCl] (2.5 mM), cereulide was much more potent. Thus, valinomycin needed 20-30 mM KCl for substantial effects, cereulide only 1-3 mM K+, which is close to its level in blood serum. This explains the higher toxicity of cereulide at low concentrations with the positively charged potassium complex being accumulated in the cell by transport through the plasma membrane driven by the membrane potential. Furthermore, with similar concentrations, the final concentration of cereulide in the cells may become higher than that of valinomycin.
Abstract: Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 microg dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (Delta Psi(m)) and the plasma membrane (Delta Psi(p)) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K(+):Na(+):Ca(2+) of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.
Abstract: Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.
Abstract: Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.
Abstract: The lipophilic toxin, cereulide, emitted by emetic food poisoning causing strains of Bacillus cereus, is a powerful mitochondria toxin. It is highly lipophilic and rapidly absorbed from the gut into the bloodstream. We tested how this toxin influences natural killer (NK) cells, which are important effectors in defence against infections and malignancy. Cereulide inhibited cytotoxicity and cytokine production of natural killer cells, caused swelling of natural killer cell mitochondria, and eventually induced natural killer cell apoptosis. The suppressive effect on cytotoxicity was fast and toxic concentration low, 20-30 microg/l. As the emesis causing concentration of cereulide is around 10 microg/kg of total body mass, our results suggest that emesis causing or even lower doses of cereulide may also have a systemic natural killer cell suppressive effect.
Abstract: Streptomyces griseus strains isolated from indoor dust have been shown to synthesize valinomycin. In this report, we show that human peripheral blood lymphocytes treated with small doses (30 ng ml(-1)) of pure valinomycin or high-pressure liquid chromatography-pure valinomycin from S. griseus quickly show mitochondrial swelling and reduced NK cell activity. Larger doses (>100 ng/ml(-1)) induced NK cell apoptosis within 2 days. Within 2 h, the toxin at 100 ng ml(-1) dramatically inhibited interleukin-15 (IL-15)- and IL-18-induced granulocyte-macrophage colony-stimulating factor and gamma interferon (IFN-gamma) production by NK cells. However, IFN-gamma production induced by a combination of IL-15 and IL-18 was somewhat less sensitive to valinomycin, suggesting a protective effect of the cytokine combination against valinomycin. Thus, valinomycin in very small doses may profoundly alter the immune response by reducing NK cell cytotoxicity and cytokine production.
Abstract: The taxonomic position of five actinobacterial strains isolated from dust, an animal shed, the air inside a museum and soil was investigated using a polyphasic approach. The growth characteristics were unusual for actinomycetes. Optimal growth was at temperatures ranging from 2 to 10 degrees C. After small-step adaptation (5 degrees C steps) to higher temperatures, the strains were also able to grow at 20 degrees C. Cell wall analyses revealed that the organisms showed a hitherto undescribed, new group B-type peptidoglycan [type B2beta according to Schleifer & Kandler (1972), but with lysine instead of ornithine]. All strains contained menaquinone MK-9. Mycolic acids were not detected. Diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid were detected in the polar lipid extracts. The main fatty acids were 12-methyl-tetradecanoic acid (15:0 anteiso), 12-methyl-tetradecenoic acid (15:1 anteiso), 14-methyl-pentadecanoic acid (16:0 iso) and 14-methyl-hexadecanoic acid (17:0 iso), as well as an unusual compound identified as 1,1-dimethoxy-anteiso-pentadecane (15:0 anteiso-DMA). The G+C content of DNA was approximately 71 mol%. The results of 16S rRNA gene sequence comparisons revealed that the strains represent a new lineage in the suborder Micrococcineae and the family Microbacteriaceae of the order Actinomycetales. On the basis of these results the new genus Frigoribacterium gen. nov. is proposed, harbouring the new species Frigoribacterium faeni sp. nov. (type strain = 801T = DSM 10309T).
Abstract: Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated. Toxins were purified from methanol extracts of the B. licheniformis biomass using boar sperm cells as the toxicity indicator. The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI-TOF-MS. The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino-acid residues L-Gln, L-Leu, D-Leu, L-Val, L-Asp, D-Leu and L-Ile in that order. Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3-hydroxy fatty acids are 13-15 carbons in length. We showed that the toxins from food and food poisoning isolates of B. licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa. Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity.
Abstract: The emetic toxin of Bacillus cereus, found to cause immobilization of spermatozoa and swelling of their mitochondria, was purified and its structure found to be identical to the earlier known toxin cereulide. It increased the conductance in black-lipid membranes in KCl solutions in an ionophore-like manner. It formed adducts with K+, Na+, and NH4+ but the conductance was highly selective for K+ in relation to Na+ and H+ (three orders of magnitude). The increase in the kinetics of conductance indicated a stoichiometric ratio between the cereulide and K+. Its ionophoretic properties are thus similar to those of valinomycin. In addition, its effects on rat liver mitochondria were similar: it stimulated swelling and respiration in respiring mitochondria in the presence but not in the absence of K+, it reduced the transmembrane potential under these conditions. In nonrespiring mitochondria, swelling was seen in KNO3- but not in NaNO3-containing media, less in acetate. In NaNO3 media addition of the cereulide caused a transient diffusion potential which was reduced by adding K+. It is concluded that the toxic effects of cereulide are due to it being a K+ ionophore.
Abstract: Stainless steel samples (AISI 316) were ennobled in a laboratory simulator with natural Baltic Sea water. After completion of ennoblement (increase of open circuit potential of ca 400 mV), the biofilm on the steel surface was characterized using confocal laser scanning microscopy (CLSM) in combination with functional and phylogenetic stains. The biofilm consisted of microbial cell clusters covering 10-20% of the surface. The clusters were loaf-formed, with a basal diameter of 20-150 wm, 5-20 per mmm2, each holding >104 cells in a density of 1-5 2 107 cells mmm3. The typical cluster contained mainly small Gram-negative bacteria (binding the EUB338 probe when hybridized in situ on the steel surface), and often carried one to three spherical colonies, either homogeneously composed of large Gram-negative cocci or more often small bacterial rods in high density, 108-109 cells mmm3. The clusters in live biofilms contained no pores, and clusters over 25 wm in diameter had a core nonpenetrable to fluorescent nucleic acid stains and ConA lectin stain. Fluorescently-tagged ConA stained cells at a depth of <5 wm, indicating the presence of cells with f-d-mannosyl and f-d-glucosyl residues on surfaces. ethidium bromide (log Kow m0.38) penetrated deeper (17 wm in 15 min, corresponding to >10 cells in a stack) into the cluster than did the less polar dyes SYTO 16 (log Kow 1.48) and acridine orange (log Kow 1.24), which stained five cells in a stack. Fluorescent hydrophobic and hydrophilic latex beads (diameter 0.02, 0.1 or 1.0 wm) coated patchwise the cluster surface facing the water, but penetrated only to depths of r2 wm indicating a permeability barrier. About 1/3 of the stainable cells hybridized in situ with Alf1b, while fewer than 1/7 hybridized to GAM42, probes targeted towards f- and n-Proteobacteria, respectively. Our results represent a microscopic description of an ennobling biofilm, where the ennoblement could follow the sequence of redox events as suggested by the model of Dickinson and Lewandowski (1996) for the structure of corrosive biofilms on a steel surface.
Abstract: Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen-1. This amount corresponds to 10(4) to 10(5) CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10(8) CFU ml-1. The detection limit for food was 3 g of rice containing 10(6) to 10(7) CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen-1, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.
Abstract: Actinomycete isolates from indoor air and dust in water-damaged schools and children's day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC50) were 10 to 63 ng (dry weight) ml of extended boar semen-1. The four strains were identified as Streptomyces griseus strains by 16S ribosomal DNA and chemotaxonomic methods. The four S. griseus strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of >/=5,000 to 72,000 ng ml-1. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of S. griseus. The EC50 of purified toxin from the S. griseus strains were 1 to 3 ng ml of extended boar semen-1, and the EC50 of commercial valinomycin was 2 ng ml of extended boar semen-1. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as S. griseus.
Abstract: Environmental properties of organic matter contained halogen and sulfur were studied in sediments of bleached kraft pulp mill effluent (BKME) recipient lakes and 2 m³ outdoor enclosures (mesocosms). The BKME contributed to 1% (v/v) of the total water flow in the lake downstream of the pulp mill where the sediments contained 1.7 to 4 mg of tetrahydrofuran extractable organic halogen (EOX-Cl) and 0.6 to 0.8 mg of tetrahydrofuran extractable organic sulfur (EOS-S) g-1 of organic matter. Upstream sediment contained 0.03 mg of EOX-Cl and 0.7 mg of EOS-S g-1 of organic matter. EOX was a better indicator for the influence of BKME in the recipient sediment than EOS. The polarity of BKME contained EOX corresponded to log Kow of < 1, and that of the downstream sediment contained EOX to > 4.5. HP-SEC analysis of the molecular weight distribution (MWD) of the EOX showed a peak between 300 to 600 g mol-1 for the BKME and between 1000 to 2000 g mol-1 for the downstream sediment. The MWD of the BKME contained EOS peaked at 300 to 1000 g mol-1, and that of the downstream sediment contained EOS at 1000 to 5000 g mol-1. These results indicate that BKME contained organic halogen and sulfur undergo major structural transformations when incorporated into sediment. The biota-to-sediment accumulation factor (BSAF) of EOX from sediments formed downstream of the mill and in the mesocosms to the lipids of Lumbriculus variegatus was 0.4 to 0.7. This is of a similar order of magnitude to the BSAF reported for 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran.
Abstract: We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable.
Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method.
One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound.
The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC.
The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively.
Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.
