Toxic indole alkaloids avrainvillamide and stephacidin B produced by a biocide tolerant indoor mold Aspergillus westerdijkiae.

Toxic Aspergillus westerdijkiae were present in house dust and indoor air fall-out from a residence and a kindergarten where the occupants suffered from building related ill health. The A. westerdijkiae isolates produced indole alkaloids avrainvillamide (445 Da) and its dimer stephacidin B (890 Da). It grew and sporulated in presence of high concentrations of boron or polyguanidine (PHMB, PHMG) based antimicrobial biocides used to remediate mold infested buildings. The boar sperm cells were used as sensor cells to purify toxins from HPLC fractions of the fungal biomass. Submicromolar concentrations (EC50 0.3-0.4 µM) blocked boar spermatozoan motility and killed porcine kidney tubular epithelial cells (PK-15). Plate grown hyphal mass of the A. westerdijkiae isolates contained 300-750 ng of avrainvillamide and 30-300 ng of stephacidin B per mg (wet weight). The toxins induced rapid (30 min) loss of boar sperm motility, followed (24 h) by loss of mitochondrial membrane potential (ΔΨm). Apoptotic cell death was observed in PK-15 cell monolayers, prior to cessation of glucose uptake or loss of ΔΨm. Avrainvillamide and stephacidin B were 100-fold more potent towards the porcine cells than the mycotoxins stephacidin A, ochratoxin A, sterigmatocystin and citrinin. The high toxicity of stephacidin B indicates a role of nitrone group in the mechanism of toxicity. Avrainvillamide and stephacidin B represent a new class of toxins with possible a threat to human health in buildings. Furthermore, the use of biocides highly enhanced the growth of toxigenic A. westerdijkiae.

Potato crop as a source of emetic Bacillus cereus and cereulide-induced mammalian cell toxicity.

Bacillus cereus, aseptically isolated from potato tubers, were screened for cereulide production and for toxicity on human and other mammalian cells. The cereulide-producing isolates grew slowly, the colonies remained small (~1 mm), tested negative for starch hydrolysis, and varied in productivity from 1 to 100 ng of cereulide mg (wet weight)(-1) (~0.01 to 1 ng per 10(5) CFU). By DNA-fingerprint analysis, the isolates matched B. cereus F5881/94, connected to human food-borne illness, but were distinct from cereulide-producing endophytes of spruce tree (Picea abies). Exposure to cell extracts (1 to 10 µg of bacterial biomass ml(-1)) and to purified cereulide (0.4 to 7 ng ml(-1)) from the potato isolates caused mitochondrial depolarization (loss of ΔΨm) in human peripheral blood mononuclear cells (PBMC) and keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine fibroblasts (L-929), and pancreatic insulin-producing cells (MIN-6). Cereulide (10 to 20 ng ml(-1)) exposed pancreatic islets (MIN-6) disintegrated into small pyknotic cells, followed by necrotic death. Necrotic death in other test cells was observed only after a 2-log-higher exposure. Exposure to 30 to 60 ng of cereulide ml(-1) induced K(+) translocation in intact, live PBMC, keratinocytes, and sperm cells within seconds of exposure, depleting 2 to 10% of the cellular K(+) stores within 10 min. The ability of cereulide to transfer K(+) ions across biological membranes may benefit the producer bacterium in K(+)-deficient environments such as extracellular spaces inside plant tissue but is a pathogenic trait when in contact with mammalian cells.

20-Residue and 11-residue peptaibols from the fungus Trichoderma longibrachiatum are synergistic in forming Na+/K+ -permeable channels and adverse action towards mammalian cells.

Certain species of the filamentous fungal genus Trichoderma (e.g. Trichoderma longibrachiatum and Trichoderma citrinoviride) are among the emerging clinical pathogens and also the most common species in the indoor space of mould-damaged buildings. The molecules involved in its pathology are not known. In the present study, we report that 0.5-2.6 wt% of the T. longibrachiatum mycelial biomass consisted of thermostable secondary metabolites mitochondriotoxic to mammalian cells. These were identified by LC/MS as one 11-residue and eight 20-residue peptaibols, AcAib-Asn-Leu/Ile-Leu/Ile-Aib-Pro-Leu/Ile-Leu/Ile-Aib-Pro-Leuol/Ileol (1175 Da) and AcAib-Ala-Aib-Ala-Aib-Ala/Aib-Gln-Aib-Val/Iva-Aib-Gly-Leu/Ile-Aib-Pro-Val/Iva-Aib-Val/Iva/Aib-Gln/Glu-Gln-Pheol(1936-1965 Da) (Aib, α-aminoisobutyric acid; Ac, acetyl; Ileol, isoleucinol; Iva, isovaline; Leuol, leucinol; Pheol, phenylalaninol). The toxic effects on boar sperm cells depended on these peptaibols, named trilongins. The trilongins formed voltage dependent, Na(+)/K(+) permeable channels in biomembranes. The permeability ratios for Na(+) ions, relative to K(+), of the 11-residue trilongin channel (0.95 : 1) and the 20-residue trilongin channel (0.8 : 1) were higher than those of alamethicin. The combined 11-residue and 20-residue trilongins generated channels that remained in an open state for a longer time than those formed by either one of the peptaibols alone. Corresponding synergy was observed in toxicokinetics. With 11-residue and 20-residue trilongins combined 1 : 2 w/w, an effective median concentration (EC(50) ) of 0.6 µg·mL(-1) was reached within 30 min, and the EC(50) shifted down to 0.2 µg·mL(-1) upon extended exposure. By contrast, with 11-residue or 20-residue trilonging separately in 30 min of exposure, the EC(50) values were 15 and 3 µg·mL(-1) , respectively, and shifted down to 1.5 and 0.4 µg·mL(-1) upon extended exposure. This is the first report on ion-channel forming peptaibols with synergistic toxicity from T. longibrachiatum strains isolated from clinical samples.

The Fusarium mycotoxins enniatins and beauvericin cause mitochondrial dysfunction by affecting the mitochondrial volume regulation, oxidative phosphorylation and ion homeostasis.

The mechanisms of cell toxicity of mycotoxins of the enniatin family produced by Fusarium sp. enniatin B, a mixture of enniatin homologues (3% A, 20% A(1), 19% B, 54% B1) and beauvericin, were investigated. In isolated rat liver mitochondria, exposure to submicromolar concentrations of the enniatin mycotoxins depleted the mitochondrial transmembrane potential, uncoupled oxidative phosphorylation, induced mitochondrial swelling and decreased calcium retention capacity of the mitochondria. The mitochondrial effects were strongly connected with the potassium (K(+)) ionophoric activity of the enniatins. The observed enniatins induced K(+) uptake by mitochondria. This shows that the enniatins acted as ionophores highly selective for potassium ions. The effects were observed in potassium containing media whereas less or no effect remained to be observed when K(+) was partially or totally replaced by isomolar concentrations of Na(+). The rank order of enniatin induced mitochondrial impairment was beauvericin>enniatin mixture>enniatin B. Exposure to the enniatins depleted the mitochondrial membrane potential also in intact human neural (Paju), murine insulinoma (Min-6) cells as well as boar spermatozoa. Exposure to enniatin B in media with physiological (4mM) or low (<1mM) but not in high (60mM) external concentration of K(+) induced hyperpolarization of the spermatozoal plasma membrane indicating enniatin that catalysed efflux of the cytosolic K(+) ions. These results indicate that the cellular toxicity targets of the enniatin mycotoxins are the mitochondrion and the homeostasis of potassium ions.

Microbial toxin's effect on mitochondrial survival by increasing K+ uptake.

We studied the effects of toxins, which inhibited the motility of boar spermatozoa, on rat liver mitochondria. The toxins studied were originally from bacteria isolated from moisture-damaged buildings where inhabitants exhibited symptoms, or from food causing poisoning. Some strains of Bacillus cereus and Streptomyces griseus produced potassium ionophoric peptides cereulide and valinomycin (Mikkola, et al., European Journal of Biochemistry 1999; 263: 112-117). Of interest is that channels were formed in black-lipid membranes (BLM) with a selectivity of K(+) > Na(+) at a concentration of 26 nM. Recently, bafilomycin A1--an inhibitor of V-H(+)ATPases--was found also to be a K(+)-specific ionophore active at nanomolar concentrations (Teplova, et al., J Bioenerg Biomembr 2007; 39: 321-329), while B. amyloliquefaciens produced amylosin, a cation channel-forming peptide with a higher selectivity for K(+) over Na(+) at around 200 nM concentrations (Mikkola, et al., Toxicon 2007; 49: 1158-1171). Of interest is that channels were formed in BLM with a selectivity of K(+) > Na(+) at a concentration of 26 nM. The ionophores and the channel-forming amylosin caused swelling of energized mitochondria due to uptake of K(+), loss of membrane potential, inhibition of maximal respiration rates due to loss of pyridine nucleotides, and inhibition of ATP synthesis. Various cell types may have different sensitivities to the effects of the ionophores. Thus, the mitochondrial membrane potential in neuronal cells was more sensitive to cereulide than in differentiated Paju cells (Teplova, et al., Acta Biochimica Polonica 2004; 51: 539-544). Swelling causes release of proapoptotic factors from mitochondria, which explains that undifferentiated neuronal cells were sensitive, while differentiated Paju cells were resistant, which probably is due to them having an increased expression of the antiapoptotic protein Bcl-2 and the neuroprotective stanniocalcin.

Novel mycotoxin from Acremonium exuviarum is a powerful inhibitor of the mitochondrial respiratory chain complex III.

A novel mycotoxin named acrebol, consisting of two closely similar peptaibols (1726 and 1740 Da), was isolated from an indoor strain of the mitosporic ascomycete fungus Acremonium exuviarum. This paper describes the unique mitochondrial toxicity of acrebol, not earlier described for any peptaibol. Acrebol inhibited complex III of the respiratory chain of isolated rat liver mitochondria (1 mg of protein mL(-1)) with an IC(50) of approximately 80 ng mL(-1) (50 nM) after a short preincubation, and 350 ng mL(-1) caused immediate and complete inhibition. Acrebol thus is a complex III inhibitor almost as potent as antimycin A and myxothiazol but completely different in structure. Similarly to myxothiazol but in contrast to antimycin A, acrebol decreased the level of mitochondrial superoxide anion detectable by chemiluminescent probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one. Unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria, did not induce disturbance of the ionic homeostasis or the osmotic balance of mitochondria, and did not release apoptogenic proteins like cytochrome c from the intermembrane space of mitochondria. In boar spermatozoa, acrebol inhibited the respiratory chain and caused ATP depletion by activation of the oligomycin-sensitive F(0)F(1)-ATPase, which resulted in the inhibition of the progressive movement. In mouse insulinoma MIN-6 cells, whose energy supply solely depends on oxidative phosphorylation, acrebol induced necrosis-like death. The pathophysiological relevance of these findings is discussed.

In vitro toxicity of cereulide on porcine pancreatic Langerhans islets.

Cereulide is a K(+) ionophore cytotoxic and mitochondriotoxic to primary cells and cell lines of human and other mammalian origins. It is a heat-stable, highly lipophilic (logK(ow) 5.96) peptide (1152 g mol(-1)) produced by certain strains of Bacillus cereus, a bacterium connected to emetic food poisonings. In this study the pancreatic toxicity of purified cereulide, and cereulide-containing bacterial extracts, was studied using fetal porcine Langerhans islets in culture. Exposure to 1ngml(-1) of purified cereulide caused necrotic cell death of the islet cells impairing their insulin content within 2 days. Cell extracts of cereulide-positive B. cereus strains connected to food poisoning or isolated from foodstuffs were toxic, corresponding to their measured cereulide content. Extracts of B. cereus strains producing or not producing the B. cereus diarrheal toxin, but no cereulide, were tolerated by the porcine islet cultures up to concentrations 1000-fold higher compared to extracts from strains containing cereulide, and up to exposure times of 7d. Cereulide thus was identified as the B. cereus-produced substance toxic towards porcine fetal Langerhans islets and beta cells.

Architecture of Deinococcus geothermalis biofilms on glass and steel: a lectin study.

Deinococcus geothermalis is resistant to chemical and physical stressors and forms tenuous biofilms in paper industry. The architecture of its biofilms growing on glass and on stainless acid proof steel was studied with confocal laser scanning microscopy and fluorescent lectins and nanobeads as in situ probes. Hydrophobic nanobeads adhered to the biofilms but did not penetrate to biofilm interior. In contrast, the biofilms were readily permeable towards many different lectins. A skeletal network of glycoconjugates, reactive with Dolichos biflorus and Maclura pomifera lectins, was prominent in the space inside the biofilm colony core but absent on the exterior. Cells in the core space of the biofilm were interconnected by a network of adhesion structures, reactive with Amaranthus caudatus lectin but with none of the 65 other tested lectins. The glycoconjugates connecting the individual cells to steel reacted with Phaseolus vulgaris lectin whereas those connecting to glass mainly reacted with A. caudatus lectin. Envelopes of all cells in the D. geothermalis biofilm reacted with several other lectins, with many different specificities. We conclude that numerous different glycoconjugates are involved in the adhesion and biofilm formation of D. geothermalis, possibly contributing its unique survival capacity when exposed to dehydration, biocidal chemicals and other extreme conditions.

Bafilomycin A1 is a potassium ionophore that impairs mitochondrial functions.

Novel activities of bafilomycin A1, a macrolide antibiotic known as an inhibitor of V-ATPases, were discovered. Bafilomycin A1 induced uptake of potassium ions by energized mitochondria and caused mitochondrial swelling, loss of membrane potential, uncoupling of oxidative phosphorylation, inhibition of the maximal respiration rates, and induced pyridine nucleotide oxidation. The mitochondrial effects provoked by nanomolar concentrations of bafilomycin A1 were connected to its activity as a potent, K(+)-specific ionophore. The K(+) ionophoric activity of bafilomycin A1 was observed also in black lipid membranes, indicating that it was an inherent property of the bafilomycin A1 molecule. It was found that bafilomycin A1 is a K(+) carrier but not a channel former. Bafilomycin A1 is the first and currently unique macrolide antibiotic with K(+) ionophoric properties. The novel properties of bafilomycin A1 may explain some of the biological effects of this plecomacrolide antibiotic, independent of V-ATPase inhibition.

Amylosin from Bacillus amyloliquefaciens, a K+ and Na+ channel-forming toxic peptide containing a polyene structure.

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.

Isolation and automated ribotyping of Mycobacterium lentiflavum from drinking water distribution system and clinical specimens.

Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.

Potential of selected infant food formulas for production of Bacillus cereus emetic toxin, cereulide.

Cereulide producing Bacillus cereus was isolated from randomly chosen commercial infant foods. The cereulide production in infant food formulas was investigated. When the reconstituted foods were inoculated with >10(5) cfu ml(-1) of cereulide producing B. cereus, 2 to 200 microg of cereulide per 100 ml of food accumulated during 24 h of non-refrigerated storage. The amount of cereulide measured in the foods by the accurate chemical assay (LC-MS) matched with that found by sperm micro assay, proving the cereulide was the sole heat stable toxin in the foods and present in its toxic form. The infant formulas containing both cereal and dairy ingredients were the most supportive for cereulide production. Cereulide accumulation was affected by the infant food composition as well as by the handling of the food. Diluting the reconstituted food with water resulted in increased toxin production expressed as mug per volume. More cereulide was accumulated when the food was incubated stationary compared with moderate shaking. The amount of cereulide accumulated within 24 h at room temperature per 100 ml of cereal and dairy or in rice-nondairy reconstituted infant formulas, inoculated with >or=10(5) cfu ml(-1) of B. cereus strain F4810/72, was higher or similar to the amounts reported for foods implicated in emetic type of food poisonings. Thus mishandling and temperature abuse of infant foods may cause food poisoning when emetic B. cereus is present.

The higher toxicity of cereulide relative to valinomycin is due to its higher affinity for potassium at physiological plasma concentration.

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.

Intranasal exposure to Stachybotrys chartarum enhances airway inflammation in allergic mice.

RATIONALE: Exposure to building dampness, often associated with growth of microbes such as Stachybotrys chartarum, has been linked to respiratory symptoms. We have shown previously in a murine model that exposure to S. chartarum can induce lung inflammation characterized by infiltration of neutrophils and lymphocytes; this process is regulated by proinflammatory cytokines and leucocyte-attracting chemokines. OBJECTIVES: Because an atopic predisposition may influence the response to microbes, we examined the effects of S. chartarum on allergic mice in an experimental model. BALB/c mice were sensitized to ovalbumin by intraperitoneal injections and exposed for 3 wk to spores of S. chartarum. MEASUREMENTS AND MAIN RESULTS: Numbers of eosinophils and neutrophils were drastically increased in bronchoalveolar fluid from these mice as compared with the ovalbumin-sensitized/challenged mice or those exposed to S. chartarum without ovalbumin sensitization. Histologic sections showed severe granulomatous inflammatory cell infiltrates in all compartments of the lung, including peribronchial, perivascular, and alveolar spaces. The mRNA levels of proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha and the chemokine CCL3/MIP-1alpha were also markedly increased in the lungs. Despite the enhancement of the pulmonary inflammatory reaction, exposure to S. chartarum spores significantly down-regulated airway hyperresponsiveness and showed a tendency to decrease levels of Th2 cytokines in the lung. CONCLUSION: Exposure to S. chartarum modulates the inflammatory reaction and airway hyperresponsiveness, depending on the allergic status of the exposed mice.

Cereulide-producing strains of Bacillus cereus show diversity.

Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600-1,800 ng cereulide mg(-1) wet weight biomass), seven average producers (180-600 ng cereulide mg(-1) wet weight biomass), four low cereulide producers (20-160 ng cereulide mg(-1) wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234-02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.

Valinomycin-induced apoptosis of human NK cells is predominantly caspase independent.

Human NK cells are sensitive to the exogenous toxic compound valinomycin. This toxin, produced by Streptomyces griseus in moisture damaged buildings, induces apoptosis by dissipating the membrane potential in mitochondria. In this paper, we show that valinomycin-induced apoptosis involves two different pathways in human NK cells: the predominant one is caspase-3 independent and the other caspase-3 dependent. Resting human NK cells were found to contain high amounts of active caspase-3 as compared to the T cells in which high caspase-3 activity has been shown only after stimulation. Exposure to valinomycin did not alter the caspase-3 activity of human NK cells but induced nucleosomal fragmentation of DNA. General caspase inhibitor, Z-VAD-FMK, inhibited completely the caspase-3 activity, reduced DNA cleavage but did not prevent the spontaneous or valinomycin-induced apoptosis of NK cells. The endogenous high caspase-3 had only a slight effect on the major functions of human NK cells, i.e. cytotoxicity or gamma-IFN production, giving us a reason to suspect that the biological role of caspase-3 in NK cells could be the elimination of potentially harmful NK clones through apoptosis.

Lipopolysaccharides of anaerobic beer spoilage bacteria of the genus Pectinatus--lipopolysaccharides of a Gram-positive genus.

Bacteria of the genus Pectinatus emerged during the seventies as contaminants and spoilage organisms in packaged beer. This genus comprises two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis; both are strict anaerobes. On the basis of genomic properties the genus is placed among low GC Gram-positive bacteria (phylum Firmicutes, class Clostridia, order Clostridiales, family Acidaminococcaceae). Despite this assignment, Pectinatus bacteria possess an outer membrane and lipopolysaccharide (LPS) typical of Gram-negative bacteria. The present review compiles the structural and compositional studies performed on Pectinatus LPS. These lipopolysaccharides exhibit extensive heterogeneity, i.e. several macromolecularly and structurally distinct LPS molecules are produced by each strain. Whereas heterogeneity is a common property in lipopolysaccharides, Pectinatus LPS have been shown to contain exceptional carbohydrate structures, consisting of a fairly conserved core region that carries a large non-repetitive saccharide that probably replaces the O-specific chain. Such structures represent a novel architectural principle of the LPS molecule.

Methylobacillus pratensis sp. nov., a novel non-pigmented, aerobic, obligately methylotrophic bacterium isolated from meadow grass.

Strain F31T was isolated from meadow grass (Poa trivialis L.) sampled from the city park in Helsinki. Analysis of phenotypic and genotypic properties showed the strain to be related to the group of obligately methylotrophic non-methane utilizing bacteria (methylobacteria) with the ribulose monophosphate pathway of formaldehyde assimilation. Phylogenetic analysis showed the strain to be closely related to the genus Methylobacillus, and analysis of fatty acid composition confirmed this association. Thus, on the basis of its genotypic and phenotypic properties, the isolate is proposed as a novel species of the genus Methylobacillus, Methylobacillus pratensis sp. nov., with F31T as the type strain (= VKM B-2247T = NCIMB 13994T).

Biological effects of Trichoderma harzianum peptaibols on mammalian cells.

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.

Sperm bioassay for rapid detection of cereulide-producing Bacillus cereus in food and related environments.

A novel in vitro method, sperm micro assay for rapidly distinguishing cereulide, the emetic toxin producing Bacillus cereus from non-producers is described and its use for quantitating cereulide and screening large numbers of B. cereus strains/colonies evaluated. The assay is non-laborious and can be executed with equipment present in most laboratories. Boar spermatozoa, purchased as standard semen from artificial insemination suppliers, are used to detect toxicity. Boar sperms respond within 5 min by cessation of motility when exposed at 37 degrees C to heat-treated (100 degrees C) extract prepared from a cereulide containing B. cereus. The assay can be done on individual colonies on the primary plate, with no need for pure culture and the qualitative result is obtained within 30 min. The assay is robust, not sensitive to age or storage of the culture plates. The use of the sperm micro assay for semiquantitative estimation of cereulide in B. cereus was validated with 14 different B. cereus strains using as reference the specific chemical assay for cereulide, based on liquid chromatography-ion trap mass spectrometry (LC-ion trap MS). The cereulide contents calculated from endpoint dilutions of the sperm micro assay matched the result of the chemical analysis closely. The detection threshold of the sperm micro assay was measured as 0.3 +/- 0.1 ng of cereulide per 5.4 x 10(6) sperm cells in 0.2 ml or 0.9 ng of cereulide per mg of B. cereus biomass (wet wt.). Food-related B. cereus strains contained 4-400 ng of cereulide per mg (wet wt.). When a large number of B. cereus of food, non-food, clinical and environmental origins were screened and 107 independent strains/isolates were identified as cereulide producers, it was observed that all of these had low or no haemolytic activity when cultivated on bovine blood agar. None of the strains/isolates with wide, clear zones of haemolysis, considered typical of B. cereus, produced cereulide.