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The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to [lambda] phage. However, contrary to the [lambda] superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage [PHI][24.sub.B]. Here, the [PHI][24.sub.B] integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the [PHI][24.sub.B] integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A [PHI][24.sub.B] gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a [PHI][24.sub.B] E. coli lysogen, and expression of an anti-repressor is the likely explanation 0or the absence of immunity to superinfection. Demonstration of the ability of [PHI][24.sub.B] to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.

A specific and sensitive quantitative real-time PCR (qPCR) method is developed for targeting the hepatotoxin nodularin synthetase gene subunit F (ndaF). This method is used to detect the ndaF containing genomes of nodularin-producing Nodularia cyanobacteria of the Baltic Sea.

A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals, such as elevated C[O.sub.2], and the trans-acting positive regulator AtxA. In addition to these requirements, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. During the late exponential phase of growth, when AbrB levels begin to decrease, toxin synthesis increases. Here we report that toxin gene expression also requires the presence of sigH, a gene encoding the RNA polymerase sigma factor associated with development in Bacillus subtilis. In the well-studied B. subtilis system, [[sigma].sup.H] is required for sporulation and other post-exponential-phase processes and is part of a feedback control pathway for abrB expression. Our data indicate that a Bacillus anthracis sigH-null mutant is asporogenous and toxin deficient. Yet the sigma factor is required for toxin gene expression in a manner that is independent of the pathway leading to post-exponential-phase gene expression. [[sigma].sup.H] positively controls atxA in an AbrB-independent manner. These findings, combined with previous observations, suggest that the steady-state level of atxA expression is critical for optimal toxin gene transcription. We propose a model whereby, under toxin-inducing growth conditions, control of toxin gene expression is fine-tuned by the independent effects of [[sigma].sup.H] and AbrB on the expression of atxA.

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, secretes several 'accessory' toxins, including RTX toxin, which causes the cross-linking of the actin cytoskeleton. RTX toxin is exported to the extracellular milieu by an atypical type I secretion system (T1SS), and we previously noted that RTXassociated activity is detectable only in supernatant fluids from log phase cultures. Here, we investigate the mechanisms for regulating RTX toxin activity in supernatant fluids. We find that exported proteases are capable of destroying RTX activity and may therefore play a role in the growth phase regulation of toxin activity. We determined that the absence of RTX toxin in stationary-phase culture supernatant fluids is also due to a lack of toxin secretion and not attributable to solely proteolytic degradation. We ascertained that the T1SS apparatus is regulated at the transcriptional level by growth phase control that is independent of quorum sensing, unlike other virulence factors of V. cholerae. Additionally, in stationary-phase cultures, all RTX toxin activity is associated with bacterial membranes or outer membrane vesicles.

ABSTRACT Outbreaks of lipophilic toxins have been recorded in the north Atlantic coast of Morocco since 1999, but are rare in the Mediterranean coast. Samples of mussels from the Atlantic [...]

The isolation and in vitro maturation of a chicken single-chain antibody variable region (scFv) directed against the low-molecular-mass (312-Da) algal toxin domoic acid (DA), its convenient production and purification and application in the development of a simple competitive enzyme immunoassay for detection of the toxin in shellfish extracts are described. The study has showed that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases. The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B. cereus strain ATCC 14579. In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity. The global regulator PlcR in B. cereus controls several putative virulence factors. It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined. This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected. It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site.

The killer toxin secreted by Kluyveromyces phaffii (KpKt) is active against spoilage yeast under winemaking conditions and thus has potential applications in the biocontrol of undesired micro-organisms in the wine industry. Biochemical characterization and N-terminal sequencing of the purified toxin show that KpKt is a glycosylated protein with a molecular mass of 33 kDa. Moreover, it shows 93% and 80% identity to a [beta]-1,3-glucanase of Saccharomyces cerevisiae and a [beta]-1,3-glucan transferase of Candida albicans, respectively, and it is active on laminarin and glucan, thus showing a [beta]-glucanase activity. Competitive inhibition of killer activity by cell-wall polysaccharides suggests that glucan ([beta-1,3 and [beta]-1,6 branched glucans) represents the first receptor site of the toxin on the envelope of the sensitive target. Flow cytometry analysis of the sensitive target after treatment with KpKt and K1 toxin of S. cerevisiae, known to cause loss of cell viability via formation of pores in the cell membrane, suggests a different mode of action for KpKt.

The use of Pichia membranifaciens CYC 1106 killer toxin against Botrytis cinerea was investigated. This strain exerted a broad-specificity killing action against other yeasts and fungi. At pH 4, optimal killer activity was observed at temperatures up to 20 [degrees]C. At 25 [degrees]C the toxic effect was reduced to 70%. The killer activity was higher in acidic medium. Above about pH 4-5 activity decreased sharply and was barely noticeable at pH 6. The killer toxin protein from P. membranifaciens CYC 1106 was purified to electrophoretic homogeneity. SDS-PAGE of the purified killer protein indicated an apparent molecular mass of 18 kDa. Killer toxin production was stimulated in the presence of non-ionic detergents. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a strain of B. cinerea. The symptoms of infection and grey mould observed in Vitis vinifera plants treated with B. cinerea were prevented in the presence of purified P. membranifaciens killer toxin. The results obtained suggest that P. membranifaciens CYC 1106 killer toxin is of potential use in the biocontrol of B. cinerea.

Pathogenic microbes can devastate populations of marine plants and animals. Yet, many sessile organisms such as seaweeds and sponges suffer remarkably low levels of microbial infection, despite lacking cell-based immune systems. Antimicrobial defenses of marine organisms are largely uncharacterized, although from a small number of studies it appears that chemical defenses may improve host resistance. In this study, we asked whether the common seaweed Lobophora variegata is chemically defended against potentially deleterious microorganisms. Using bioassay-guided fractionation, we isolated and characterized a 22-membered cyclic lactone, lobophorolide (1), of presumed polyketide origin, with sub-[micro]M activity against pathogenic and saprophytic marine fungi. Deterrent concentrations of I were found in 46 of 51 samples collected from 10 locations in the Bahamas over a 4-year period. Lobophorolide (1) is structurally unprecedented, yet parts of the molecule are related to tolytoxin, the scytophycins, and the swinholides, macrolides previously isolated from terrestrial cyanobacteria and from marine sponges and gastropods. Until now, compounds of this structural class have not been associated with marine macrophytes. Our findings suggest that seaweeds use targeted antimicrobial chemical defense strategies and that secondary metabolites important in the ecological interactions between marine macroorganisms and microorganisms could be a promising source of novel bioactive compounds.

Ever since it was blamed for massive fish kills in estuaries in the eastern United States a decade ago, a tiny one-celled organism known as Pfiesteria piscicida has fascinated and [...]

Strains of Periconia circinata that are not pathogenic appear to be deficient in the synthesis of peritoxins. This was the conclusion of researchers who found intermediates of peritoxin synthesis only in pathogenic, toxin-producing strains but not in non-pathogenic, non-toxin-producing strains.

The Canadian AIDS Society (CAS) wants to raise awareness of the need for topical microbicides as an HIV and other sexually transmitted diseases (STD) prevention alternative. CAS also recognizes the [...]

A killer protein isolated from the yeast Schwanniomyces occidentalis has sequence similarity to killer toxin D2 from Saccharomyces cerevisiae. This strain of S. occidentalis could be used as a starter in commercial fermentations to prevent contamination by wild killer yeasts.

Research demonstrates the structural configuration of the stereogenic centers C-7, C-8, and C-9 of pinolidoxin by stereoselective synthesis of its C-(sub)6-C(sub)18 portion and by spectral measurements. The stereoselective total synthesis of pinolidoxin using the C(sub)6-C(sub)18 portion is described.

DNA sequence analysis of a 6-kb pathogenicity island, from113 enterotoxigenic and 191 nontoxigenic Bacterioides fragalis, shows that the specific segment and its flanking regions originate from different mobile genetic elements. This indicates that the enterotoxigenic bacteria may have evolved by horizontal transfer of these two genetic elements.

A molecular plasma membrane target for K1 toxin, known to bring uncontrolled leading of potassium, protons, ATP and other small molecules from target cells in Saccharomyces cerevisiae, has been found. Killer strains of S. cerevisiae have doublestrand RNA viruses and ccrete protein toxins that kill virus-free cells. TOK1 is an outwardly rectifying, potassium-selective ion channel. The toxin activates this plasma membrane potassium channel. Channels have also been studied in Xenopus laevis oocytes.

The exfoliative toxins (ETs) cause staphylococcal scalded skin syndrome, a disease characterized by specific separation of layers of the skin. Research has shown that the toxins act as serin proteases, though the specific substrate and mode of action are not known for certain. The crystal structure of exfoliative toxin A (ETA) has been found previously to be similar to that of chymotrypsin-like serine proteases. The 2.4 angstrom resolution crystal structure of the other exfoliative toxin, ETB, which is 40% identical to ETA.

The first evidence of clear antagonism between toxins from different subspecies of Bacillus thuringiensis, detected both in vivo and in vitro, is reported. These two quite different B thuringiensis toxins are Cry1Ac1, which has a high activity toward members of the Lepidoptera, and Cyt1A1, which came from a mosquitocidal strain but has a broad activity range because of its cytolytic capacity. Data from other joint-action analyses affirm these results.

The possible role of domain III of the toxic fragment of Bacillus thuringiensis crystal proteins in toxin specificity and in midgut epithelial membrane binding specificity in the diamondback moth was explored using hybrid toxins with domain III substitutions in Cry1C, Cry1E and Cry1Ab. Results showed that domain II and/or domain I, and not domain III, mediates toxin specificity as well as specificity of binding to membrane vesicles in the diamondback moth.

Membrane vesicles released by Escherichia coli O157:H7 contain DNA encoding the virulence genes eae, stx1 and stx2, as well as uidA, which encodes for beta-galactosidase, while immunoblotting data show that Shiga toxins 1 and 2 are contained within vesicles. This was gleaned from a study that sought to determine whether E coli O157:H7 produces vesicles under normal growth conditions and whether these contain Shiga toxins and DNA and transfer virulent genes. Results indicate that these vesicles play a role in the export of toxic and genetic material. They can facilitate the transfer of genetic material to other enteric organisms and may serve to disseminate toxic material directly to host cells or to bacterial cells.

Research was conducted to investigate the existence of two new genes in Campylobacter rectus coding for bistructural surface-layer (S-layer)-RTX proteins, designated CsxA and CsxB. The two proteins were found to have common function as demonstrated by the strong relatedness observed within their amino acid sequences. Results indicate that a putative RTX homologue seen in C. rectus by means of screening with broad-range RTX gene probes represents a new class of putative bifunctional proteins having both S-layer and RTX domains.

Research was conducted to examine the toxicity, binding and permeability of four Bacillus thuringiensis Cry1 toxins using brush border membrane vesicles from Spodoptera frugiperda and Spodoptera exigua. Results provide evidence that the toxicities of the four toxins differed significantly. Findings also indicate that there was no relationship between toxicity and dissociation of bound toxins from brush border membrane vesicles.

The microorganisms which can degrade thaxtomin A, the major phytotoxin produced by Streptomyces scabies, were determined for potential use as biocontrol agents for potato scab. Findings revealed that various microorganisms including gram-positive and gram-negative bacteria and even fungi can degrade thaxtomin A. The thaxtomin A-degrading fungi were not able to control potato scab. However, all three thaxtomin A-utilizing bacteria were able to protect potato tubers from developing scabs.

Human gastric epithelial cells in culture were used to investigate the uptake, localization and fate of the Helicobacter pylori vacuolating toxin A (VacA) and the time of persistence of toxin inside the cell. Findings showed that VacA is transported to cells in both free and membrane-bound form, localizes inside the endosomal-lysosomal compartment, persists within the cell for at least 72 hours and does not degrade into smaller fragments. These suggests that a large amount of VacA avoids the main lysosomal degradative processes and retains its molecular integrity.

A case-control study was conducted to examine toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata during a study of pulmonary hemosiderosis in infants occurring in Cleveland, OH, in 1993 and 1994. The isolates from S. chartarum were shown to produce a number of highly toxic compounds and the profiles of toxic compounds from M. echinata were similar. The most distinct difference was the fact that the principal metabolites produced by M. echinata were griseofulvins.

Verotoxins are a class of bacterial toxins whose morphology constitutes an enzymatic A subunit where the toxic activity resides and is linked to a B oligomer that binds to specific cell-surface carbohydrate receptors. The solution structure of this carbohydrate B subunit of verotoxin VT-1 from the enterohemmorrhagic Escherichia coli is determined in relation to the trimethylsilylethyl derivative of the trisaccharide moiety of Gb3. The solution structure was obtained using nuclear magnetic resonance methods and is described.

Beauvericin secretion has been measured in 94 isolates belonging to 25 Fusarium taxa. Beauvericin is a cyclohexadepsipeptide mycotoxin with insecticidal qualities and induces apoptosis in mammalian cells. It is found naturally on corn and corn-based foods infection by Fusarium. Results show that beauvericin is produced by many Fusarium species such as F. acuminatum, F. anthophilum and F. avenaceum. There is also evidence that beauvericin contaminates other cereals besides corn.

Research was conducted to examine the expression of Bacillus thuringiensis (Cryl1Ac) toxin in two different phage display vector systems. Both systems successfully permit the expression of active toxin as a fusion protein on filamentous phage. Results demonstrate that activated Cryl1Ac can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. The phage display of B. thuringiensis toxins exhibits many advantages over previous expression systems for the proteins.

Experiments were performed to investigate the mechanism behind the temperature-regulation of heat-labile enterotoxin (LT) synthesis in Escherichia coli. DNA manipulation and analysis of Ent plasmids, transmissible plasmids that encode LT and heat-stable enterotoxin, showed that protein and messengerRNA levels of LT were highest at 37 degrees centigrade and established a direct relationship between protein levels and temperature. The involvement of downstream regulatory element in the temperature regulation of LT synthesis was ruled out.

Research was conducted to examine a new, sensitive, inexpensive and rapid bioassay for the detection of the emetic toxin of Bacillus cereus. The novel bioassay is based on the motility loss of boar spermatozoa upon 24 h of exposure to emetic B cereus strains extracts or contaminated food. Results indicate swollen mitochondria of the paralyzed spermatozoa but no epetion of cellular ATP or damage to plasma membrane integrity was observed.

Site-directed mutagenesis was applied to examine the mode of action of the binary toxin formed by the combination of two mosquito larvicidal proteins of 51 and 42kDa. On its own, each of these peptides is nontoxic. When synthesized by Bacillus sphaericus 1593M during sporulation, the peptides of 51 and 42 kDa show toxicity. The impact of the substitution of alanine residues for other amino acids found at the N- and C-terminal regions of the binary the binary toxin on biological activity was investigated.

A study was conducted on the binding of the haemagglutinin of type C 16S toxin to epithelial cells of the upper small intestine of guinea pigs. The findings indicate that haemogglutinin of type C 16S toxin plays an essential role in the binding and absorption of botulinum toxin in the small intestine. Further studies are recommended on the detailed absorption mechanism of the 12S toxins.

Cloning and sequencing of a cluster of genes which compose part of the general secretion pathway (GSP) of Vibrio cholerae were conducted. The results indicate that mutations in the eps genes lead to aberrant outer membrane protein profiles. This suggests that the GSP, or at least some of its parts, is required both for the secretion of soluble proteins and for proper outer membrane assembly.

A laboratory experiment was performed on cyanobacterial hepatotoxins, microcystins, to determine the significance of external growth stimuli in controling the levels and relative proportions of various microcystin variants in two strains of filamentous, nitrogen-fixing Anabaena spp. Findings indicated that the reduction of phosphorus loads in bodies of water may be used in preventing the health hazards that toxic cyanobacterial water blooms pose, not only by reducing the cyanobacteria but also by reducing their toxic content.

Laboratory selection improved the resistance to the Bacillus thuringiensis toxin Cry1C in a strain of diamondback moth Plutella xylostella. The selected strain was derived from a field population that had evolved high levels of resistance to Bacillus thuringiensis subsp. kurstaki and moderate resistance to Cry1C of the selected strain increased to 62-fold after six generations of selection. Analysis of F1 hybrid progeny from reciprocal crosses between the selected strain and susceptible strain indicated that the resistance to Cry1C was autosomally inherited.

Two separate animal populations composed of a herd of cattle and a flock of sheep were studied for strains of Escherichia coli producing Shiga toxins (STEC) for a period of six months. Thirty-three STEC were isolated from 63.2% of cattle and grouped into 11 serotypes and eight electrophoretic types by multilocus enzyme analysis. Eighty-eight percent of the sheep excreted STEC belonging to 17 different serotypes and 12 different electrophoretic types.

Three schemes proposed in the literature for analyzing joint toxic effects of multicomponent mixtures on fish, namely the additivity index (AI), the mixture toxicity index (MTI), and the similarity parameter ([Lambda]) are evaluated in this study for microbial toxicity. A new approach is proposed to establish acceptance limits for the similarity parameter, [Lambda], based on experimental errors and uncertainties. Quantitative structure activity relationship (QSAR) techniques are then used to develop a model to predict the concentrations of components in mixtures that would jointly cause 50% inhibition of microbial respiration. The application of this approach is demonstrated on the experimental toxicity data of six eight-component organic chemical mixtures on microorganisms.

The distribution of binary toxin (btx) and mosquitocidal toxin (mtx) genes in 53 strains of bacillus sphaericus was investigated by hybridization of specific gene probes to chromosomal DNA in Southern blots. btx genes were observed in all strains of serotypes 5a5b and in some strains of serotypes 1a, 3, 6, 25 and 48, while mtx genes were found in strains of serotypes 1a, 2a2b, 5a5b, 6, 9a9c, 25 and 48. Serotype 26a26b strains did not have both toxin genes.

Transposon mutagenesis of the Xanthomonas alibilineans X32R1 reveals 2 gene clusters responsible for the production of the phytotoxin albicidin. The mutants are defective in their ability to produce albicidin and span a 48kb genomic region. However, some of these mutants are still able to colonize sugarcane plants cultivated in vitro. Different Xanthomonas albicidineans strains show identical hybridization, indicating that other strains of the pathogen may also contain at least 2 genetic clusters responsible for albicidin production.

The TOXA gene of the plant-pathogen fungus, Cochliobolus carbonum, encodes an HC-toxin efflux pump which plays a role in self-protection against HC-toxin. The gene is present only in the fungal isolates that make HC-toxin, and it occurs as two linked copies. TOXA occurs close to HTS1, the gene found in HC-toxin making fungal isolates, that codes for the enzyme necessary for the synthesis of HC-toxin. The amino acid sequence of TOXA gene is discussed.

The three-dimensional structures of the cyclic cyanobacterial heptapeptide toxin microcystin-LR was characterized using nuclear magnetic resonance spectroscopy in aqueous and dimethyl sulfoxide (DMSO)/water solutions. Spatial arrangement was further studied by simulated annealing and molecular dynamics calculations. In solution, microcystin-LR predicted a single saddle-shaped backbone conformation with the flat part consisting of the residues Adda5-Glu6-Mdha7. The side segments of Arg4 and the remote portion of Adda5 are described to be pliable with no discernible structures.

The ability of mAbs produced against protective antigen (PA) to neutralize lethal toxin activity in Bacillus anthracis is compared with their ability to inhibit binding of PA to cells and of lethal factor to cell-bound PA. mAbs which perform these two binding interactions are mapped to three different regions on PA. Results indicate that mAbs are able to identify antigenic regions. However, they fail to identify the putative translocation region on PA.

The action of human enteric bacterial toxin has been tested on intestinal mucosa and intestinal epithelial cells. Diarrhea is believed to be caused by several toxin-producing enteric bacteria whose toxin stimulates intestinal epithelial secretion. Toxins are classified under three types according to the damage done to intestinal cells. A number of enteric bacteria are identified with their corresponding toxin/toxins.

A mixture of the Bacillus thuringiensis toxic crystal proteins CryIAa and CryIAc is more toxic for the gypsy moth Lymantria dispar than the individual proteins. The synergistic activity of the proteins appears to be due to the formation of a CryIAa-CryIAc hetero-oligomer that binds to the CryIAa receptor, increasing toxicity. A mixture of CryIAa and CryIAb is however, less toxic than the individual proteins due to an antagonistic effect. The antagonism is probably due to competition as both the proteins bind to the same receptor. No synergistic activity of the CryIAa-CryIAc mixture is seen in the silkworm.