Transfer and expression of the cryIVB and cryIVD genes of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus 2297

Abstract The genes encoding the CryIVB and CryIVD crystal polypeptides of B. thuringiensis subsp. israelensis were cloned indepently on a stable shuttle vector, and transfered into B. sphaericus 2297. Recombinant cells expressed the B. thuringiensis toxins during sporulation and were shown to be toxic to Aedes aegypti fourth instar larvae, whereas the parental strain was not.     

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C₉H₁₆N₆O₂. This structure differed from that of decarbamoyl STX (dcSTX; C₉H₁₆N₆O₃) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.     

Structural variability of C3larvin toxin. Intrinsic dynamics of the α/β fold of the C3-like group of mono-ADP-ribosyltransferase toxins

C3larvin toxin is a new member of the C3 class of the mono-ADP-ribosyltransferase toxin family. The C3 toxins are known to covalently modify small G-proteins, e.g. RhoA, impairing their function, and serving as virulence factors for an offending pathogen. A full-length X-ray structure of C3larvin (2.3 Å) revealed that the characteristic mixed α/β fold consists of a central β-core flanked by two helical regions. Topologically, the protein can be separated into N and C lobes, each formed by a β-sheet and an α-motif, and connected by exposed loops involved in the recognition, binding, and catalysis of the toxin/enzyme, i.e. the ADP-ribosylation turn–turn and phosphate–nicotinamide PN loops. Herein, we provide two new C3larvin X-ray structures and present a systematic study of the toxin dynamics by first analyzing the experimental variability of the X-ray data-set followed by contrasting those results with theoretical predictions based on Elastic Network Models (GNM and ANM). We identify residues that participate in the stability of the N-lobe, putative hinges at loop residues, and energy-favored deformation vectors compatible with conformational changes of the key loops and 3D-subdomains (N/C-lobes), among the X-ray structures. We analyze a larger ensemble of known C3bot1 conformations and conclude that the characteristic ‘crab-claw’ movement may be driven by the main intrinsic modes of motion. Finally, via computational simulations, we identify harmonic and anharmonic fluctuations that might define the C3larvin ‘native state.’ Implications for docking protocols are derived.     

A Survey Investigating the Infection of Fusarium langsethiae and Production of HT‐2 and T‐2 Mycotoxins in UK Oat Fields

Fusarium langsethiae is a toxigenic fungal species that has been reported in European small‐grain cereal crops such as oats, wheat and barley. Although its relative contribution to fusarium head blight (FHB) symptoms is not well understood, it is reported to contaminate these cereals with high levels of HT‐2 and T‐2 trichothecenes mycotoxins that are currently under consideration for legislation by the European Commission. Ten commercial oat fields in Shropshire and Staffordshire (two adjacent counties in the Midlands) in the UK were surveyed in the 2006/2007 growing season. Samples were taken from predetermined field locations at Zadoks growth stages 32/33, 69, 77‐85 and 90‐92 for F. langsethiae biomass and HT‐2 and T‐2 toxins quantification. The results from this study showed that oats can be heavily infected with F. langsethiae and have high concentrations of HT‐2 and T‐2 toxins with no apparent FHB symptoms. The regression of HT‐2 + T‐2 toxins on F. langsethiae DNA concentration was highly significant (P < 0.001, r² = 0.55). The results indicated that although F. langsethiae had no direct effect on crop yield, it may result in indirect economic losses where the grain can be rejected or downgraded as a result of intolerable levels of HT‐2 and T‐2 toxins, which are of human food and animal feed safety concern. The influence of cultural field practices on the infection and HT‐2 and T‐2 toxins accumulation in oats was not clear and warrants further studies to identify the sources of F. langsethiae inoculum and conditions favourable for infection and mycotoxin production.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.     

Tomato resistance to Alternaria stem canker: localization in host genotypes and functional expression compared to non-host resistance

Summary The Alternaria stem canker resistance locus (Asc-locus), involved in resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici and in insensitivity to host-specific toxins (AAL-toxins) produced by the pathogen, was genetically mapped on the tomato genome. Susceptibility and resistance were assayed by testing a segregating F₂ population for sensitivity to AAL-toxins in leaf bioassays. Linkage was observed to phenotypic markers solanifolium and sunny, both on chromosome 3. For the Asc-locus, a distance of 18 centiMorgan to solanifolium was calculated, corresponding to position 93 on chromosome 3. This map position of the resistance locus turned out to be the same in three different resistant tomato accessions, one Dutch and two American, that are at least 40 years apart. AAL-toxin sensitivity in susceptible and resistant tomato genotypes was compared with AAL-toxin sensitivity in a non-host Nicotiana tabacum during different levels of plant cell development. In susceptible and resistant tomato genotypes, inhibitory effects were demonstrated at all levels, except for leaves of resistant genotypes. However, during pollen and root development, inhibitory effects on susceptible genotypes were larger than on resistant genotypes. In the non-host Nicotiana tabacum, hardly any effects of AAL-toxins were demonstrated. Apparently, a cellular target site is present in tomato, but not in Nicotiana tabacum. It was concluded that three levels of AAL-toxin sensitivity exist: (1) a susceptible host sensitivity, (2) a resistant host sensitivity, (3) a non-host sensitivity, and that the resistance mechanism operating in tomato is different from that operating in Nicotiana tabacum.     

Photoaffinity labeling of the receptor site forα-Scorpion toxins on purified and reconstituted sodium channels by a new toxin derivative

1. A methyl-4-azidobenzimidyl (MAB) derivative of the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) specifically labels only the alpha subunit of the rat brain sodium channel in synaptosomes or in purified and reconstituted sodium-channel preparations. 2. Unlike previous photoreactive toxin derivaties, binding and photolabeling by MAB-LqTx are allosterically modulated by tetrodotoxin and batrachotoxin, as observed for native LqTx binding to sodium channels in synaptosomes. 3. Proteolytic cleavage of the alpha subunit photolabeled with MAB-LqTx shows that the label is located within a 60 to 70-kDa protease-resistant core structure in domain I of the sodium-channel alpha subunit. 4. MAB-LqTx will be valuable in further defining the structure-activity relationships at the alpha-scorpion toxin receptor site.     

Effects of Alternaria alternata f.sp. lycopersici toxins on pollen

Summary Effects of the phytotoxic compounds (AAL-toxins) isolated from cell-free culture filtrates of Alternaria alternata f.sp. lycopersici on in vitro pollen development were studied. AAL-toxins inhibited both germination and tube growth of pollen from several Lycopersicon genotypes. Pollen from susceptible genotypes, however, was more sensitive for AAL-toxins than pollen from resistant plants, while pollen of species not belonging to the host range of the fungus was not significantly affected by the tested toxin concentrations. AAL-toxins elicit symptoms in detached leaf bioassays indistinguishable from those observed on leaves of fungal infected tomato plants, and toxins play a major role in the pathogenesis. Apparently, pathogenesis-related processes and mechanisms involved in disease resistance are expressed in both vegetative and generative tissues. This overlap in gene expression between the sporophytic and gametophytic level of a plant may be advantageously utilized in plant breeding programmes. Pollen may be used to distinguish susceptible and resistant plants and to select for resistances and tolerances against phytotoxins and other selective agents.     

Cholesterol as a target for toxins

A mechanism is proposed for the way in which cholesterol facilitates channel formation by polyene antibiotics and bacterial protein toxins. Central elements of the model are: (i) interactions between the ring system of the sterol and rigid elements of the polyene or toxin molecule, and (ii) the specific orientation of cholesterol within the membrane.     

Permeability Increase Induced by Escherichia coli Hemolysin A in Human Macrophages is Due to the Formation of Ionic Pores: A Patch Clamp Characterization

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K⁺ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches. Received: 14 August 1995/Revised: 2 October 1995