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D J Ellar

Publications and source records attributed to D J Ellar.

At least 73 records · Page 4Linked to original sources
PubMed1989

Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2.

The mosquitocidal crystal of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2 was purified, bioassayed against third-instar Aedes aegypti larvae (50% lethal concentration, 7.5 micrograms/ml), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing polypeptides of 125, 50, 47, and 28 kilodaltons (kDa). When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was hemolytic. By using nondenaturing polyacrylamide gel electrophoresis, a polypeptide of 23 kDa, derived from the 28-kDa protoxin, was identified which was hemolytic and cytotoxic to Aedes albopictus, A. aegypti, and Choristoneura fumiferana CF1 insect cell lines. The 23-kDa polypeptide was purified by ion-exchange chromatography and gave 50% lethal dose values of 3.8, 3.3, and 6.9 micrograms/ml against A. albopictus, A. aegypti, and C. fumiferana CF1 cells lines, respectively. Cytotoxicity in vitro was both dose and temperature dependent, with a sigmoidal dose-response curve. The cytotoxicity of the 23-kDa toxin and the solubilized and proteolytically activated delta-endotoxin was inhibited by a range of phospholipids containing unsaturated fatty acids and by triglyceride and diglyceride dispersions. An interaction with membrane phospholipids appears important for toxicity. Polyclonal antisera prepared against the 23-kDa polypeptide did not cross-react with polypeptides in the native crystals of four other mosquitocidal strains.

Animals↗
PubMed1989

Evidence for two different types of insecticidal P2 toxins with dual specificity in Bacillus thuringiensis subspecies.

Analysis of polypeptides in the crystalline delta-endotoxins from different Bacillus thuringiensis strains revealed two antigenically similar forms of the P2 protein which differed in molecular mass, peptide profile, and amino acid sequence. Purified preparations of the two forms displayed the characteristic dual toxicity of the P2 protein towards members of the orders Lepidoptera and Diptera in vivo but differed markedly in potency for the insects tested. Both species of the P2 protoxin, solubilized and activated by sequential proteolysis with insect gut extract and alpha-chymotrypsin, retained activity in vivo and in vitro, despite the removal of 144 residues from the N terminus. For the low-molecular-mass form, the dual insecticidal activity was reproducible in the in vitro assays.

Amino Acid Sequence↗
PubMed1989

Action of activated 27,000 Mr toxin from Bacillus thuringiensis var. israelensis on Malpighian tubules of the insect, Rhodnius prolixus.

The action of activated 27,000 Mr toxin from Bacillus thuringiensis var. israelensis (Bti toxin) on Malpighian tubules of Rhodnius prolixus has been investigated. Its binding to the tubules is slowed by low temperature but is not prevented even at 0 degree C. The binding is less effective at pH 10 than at pH7. Pretreatment of the tubules with 0.1 mmol l-1 ouabain or bumetanide or 1 mumol l-1 5-hydroxytryptamine did not affect the toxicity of the toxin. The toxin causes very large changes in the trans-epithelial potential difference; it changes from 40 mV, lumen negative, often to more than 100 mV, lumen positive. This reflects an initial collapse of the potential of the basal cell membrane, followed by a large positive-going potential change at the luminal cell membrane. Just prior to the effects of the toxin on rapid fluid secretion, the basal cell membrane becomes permeable to sucrose molecules. Raffinose at 170 mmol l-1 in the bathing solution does not protect the tubules from Bti toxin action but dextran, Mr5000, at 60 mmol l-1 significantly delayed failure of fluid secretion and, even more, the onset of staining of the tubule cells with Trypan Blue. Exposing tubules to saline that is calcium-free and/or magnesium-free, or has a composition adjusted to be similar to that of the intracellular milieu, does not affect the time course of failure of fluid secretion induced by the toxin. There is no evidence that effective aggregates of Bti toxin molecules are formed in concentrated solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Single amino acid changes in the Bacillus thuringiensis var. israelensis delta-endotoxin affect the toxicity and expression of the protein.

Site-directed mutagenesis has been used to change individual amino acids of the larvicidal 27,000 Mr delta-endotoxin of Bacillus thuringiensis var. israelensis. Basic and acidic residues have been systematically replaced by alanine, and the resulting mutant polypeptides analysed for cytolytic and larvicidal activity, and binding to phosphatidyl choline liposomes. Replacement of residues at positions 154, 163, 164, 213 and 225 results in proteins which accumulate as inclusions in recombinant Bacillus subtilis cells similar to the wild-type, but have considerably reduced in-vitro and in-vivo toxicity. One mutant (Glu45 to Ala45) results in a protein that has reduced activity in vitro, but retains wild-type larvicidal toxicity. In addition, seven other mutations of charged residues result in proteins which form small or no inclusions in recombinant cells, despite being produced at levels similar to the wild-type in six out of seven cases. In most instances, the toxicity of these aberrantly expressed proteins is considerably less than the wild-type, although one (Lys124 to Ala124) results in a polypeptide with approximately threefold increased activity in vitro. A secondary structural model is proposed to explain these observations.

Amino Acid Sequence↗
PubMed1988

Comparison of the in vivo and in vitro activity of the delta-endotoxin of Bacillus thuringiensis var. morrisoni (HD-12) and two of its constituent proteins after cloning and expression in Escherichia coli.

The insecticidal crystal delta-endotoxin of Bacillus thuringiensis var. morrisoni HD-12 contains at least five polypeptides in the range 126-140 kDa. Immune blotting revealed that individual proteins in this complex share homology with a range of other B. thuringiensis delta-endotoxins. In vivo the native HD-12 crystal killed a lepidopteran larva (Pieris brassicae) and a dipteran larva (Anopheles gambiae), but not the related dipteran Aedes aegypti. In vitro the solubilized activated crystal lysed Choristoneura fumiferana cells (lepidopteran) and dipteran cells derived from Anopheles gambiae and Culex quinquefasciatus but not those from Aedes aegypti. An intragenic probe derived from a B. thuringiensis var. sotto lepidoptera-specific delta-endotoxin gene hybridized with one of six plasmids extracted from HD-12. When cloned into pUC18 two HindIII fragments from this plasmid (pEG1 and pEG2) were shown to encode polypeptides cross-reacting with HD-12 antiserum. Escherichia coli lysates containing pEG2 were toxic in vivo to lepidoptera and diptera larvae and in vitro to a broader range of insect cell lines than the native crystal. E. coli cells containing pEG3, a subclone derived from pEG1, synthesised large amounts of a 140-kDa protein in the cytoplasm as inclusion bodies. The cytotoxicity of the protein encoded by pEG3 was restricted to C. fumiferana and A. gambiae cell lines.

Animals↗
PubMed1988

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro.

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.

Aedes↗
PubMed1988

Differential specificity of two insecticidal toxins from Bacillus thuringiensis var. aizawai.

Bacillus thuringiensis var. aizawai HD-249 produces more than one protein of 130-135 kD in its insecticidal crystal delta-endotoxin. We describe an indirect method of assessing the relative contribution to toxicity of two of these protoxins using monospecific antibodies directed against their active proteolytic products. Our results show that one toxin is active against Spodoptera frugiperda but not Choristoneura fumiferana cells in vitro, while the other lyses C. fumiferana but not S. frugiperda cells. There is no indication of synergism between these toxins in vitro.

Antigen-Antibody Complex↗
PubMed1988

Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins.

Two homologous genes encoding 130-kilodalton (kDa) mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis have been cloned and expressed in Escherichia coli or Bacillus subtilis or both. One of these genes, pPC130, was expressed as a lacZ transcriptional fusion in E. coli at a level sufficient to produce phase-bright inclusions, which were purified and shown to be toxic to Aedes aegypti larvae. The second gene, pCH130, was expressed at a low level in recombinant E. coli cells and was therefore cloned in B. subtilis as a transcriptional fusion of the promoter sequences corresponding to a B. thuringiensis subsp. israelensis 27-kDa delta-endotoxin (E. S. Ward, A. R. Ridley, D. J. Ellar, and J. A. Todd, J. Mol. Biol. 191:13-22, 1986) and the structural gene. Recombinant B. subtilis cells produced phase-bright inclusions during late sporulation; these were partially purified and shown to be toxic to A. aegypti larvae at an LC50 (concentration required to cause 50% mortality of larvae after 24 h of assay) which is significantly lower than that of the pPC130 protein. Neither 130-kDa protein was hemolytic under the assay conditions. Comparison of the nucleotide sequences of these two genes indicates that they share a high degree of homology in the C-terminal portions, but relatively little similarity in the N termini. In addition, significant homologies were found between the pCH130 gene and the HD-1 Dipel gene of B. thuringiensis subsp. kurstaki (H. E. Schnepf, H. C. Wong, and H. R. Whiteley, J. Biol. Chem. 260:6264-6272, 1985).

Aedes↗
PubMed1988

Dacus oleae microbial symbionts.

Dacus oleae (Gmelin), which is a major insect pest of the olive tree, is closely associated during all developmental stages with a bacterial symbiont. This review describes the localization and transmission of the symbiont, the attempts made to isolate, cultivate and characterize it, and the host-symbiont interactions.

Animals↗
PubMed1987

Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal delta-endotoxin.

The mechanism of action and receptor binding of a dual-specificity Bacillus thuringiensis var. aizawai ICl delta-endotoxin was studied using insect cell culture. The native protoxin was labelled with 125I, proteolytically activated and the affinity of the resulting preparations for insect cell-membrane proteins was studied by blotting. The active preparations obtained by various treatments had characteristic specificity associated with unique polypeptides, and showed affinity for different membrane proteins. The lepidopteran-specific preparation (trypsin-treated protoxin containing 58 and 55 kDa polypeptides) bound to two membrane proteins in the lepidopteran cells but none in the dipteran cells. The dipteran-specific preparation (protoxin treated sequentially with trypsin and Aedes aegypti gut proteases, containing a 53 kDa polypeptide) bound to a 90 kDa membrane protein in the dipteran (A. aegypti) cells but bound to none in the lepidopteran cells or Drosophila melanogaster cells. The toxicity of trypsin-activated delta-endotoxin was completely inhibited by preincubation with D-glucose, suggesting a role for this carbohydrate in toxin-receptor interaction. The toxicity was also decreased by osmotic protectants to an extent proportional to their viscometric radius. These results support a proposal that initial interaction of toxin with a unique receptor determines the specificity of the toxin, following which cell death occurs by a mechanism of colloid osmotic lysis.

Bacillus thuringiensis Toxins↗
PubMed1987

Cloning and heterologous expression of an insecticidal delta-endotoxin gene from Bacillus thuringiensis var. aizawai IC1 toxic to both lepidoptera and diptera.

Bacillus thuringiensis var. aizawai IC1 synthesises an insecticidal protein delta-endotoxin (130-135 kDa) that is toxic to both lepidopteran and dipteran larvae, and cross-reacts immunologically with certain monospecific lepidopteran toxins. A 166-kb plasmid from this bacterium was found to hybridise with an intragenic probe derived from the clone B. thuringiensis var. sotto lepidopteran-specific delta-endotoxin gene. A strongly hybridising 5.2-kb SstI fragment from var. aizawai plasmid DNA was cloned in pUC18. After subcloning of this DNA in Escherichia coli, recombinants were obtained that synthesised large amounts of a 130-135-kDa protein. The protein was deposited in the cytoplasm as microscopically visible inclusion bodies and lysates of these cells were found to be toxic to both lepidopteran and dipteran larvae by comparison with controls. The structural basis for the dual specificity of this var. aizawai toxin is now amenable to further study.

Bacillus thuringiensis↗
PubMed1987

Cloning and expression in Escherichia coli of an insecticidal crystal protein gene from Bacillus thuringiensis var. aizawai HD-133.

Using a gene probe derived from the cloned var. sotto insecticidal crystal protein (ICP) gene, we have cloned a Bacillus thuringiensis var. aizawai HD-133 ICP gene in Escherichia coli. The gene encodes a polypeptide that is toxic to Lepidoptera in vivo and in vitro. The protein is expressed at a level sufficient to produce phase-bright inclusions in recombinant E. coli strains, and these inclusions can be partially purified using discontinuous sucrose density gradients. Immunoblotting shows that the inclusions contain a 135 kDa polypeptide which reacts strongly with antiserum raised against the B. thuringiensis var. kurstaki HD-1 P1 polypeptide.

Bacillus thuringiensis↗
PubMed1987

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture.

An insecticidal protein gene from Bacillus thuringiensis var. aizawai was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteolytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resistant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CF1 cells but not dipteran (Aedes albopictus) cells. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawai toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran cell membranes, which may be the receptor for this toxin.

Animals↗
PubMed1986

Bacillus thuringiensis var. israelensis delta-endotoxin. Nucleotide sequence and characterization of the transcripts in Bacillus thuringiensis and Escherichia coli.

The nucleotide sequence of a 1408 base-pair DNA fragment encoding the insecticidal 27,340 Mr delta-endotoxin of Bacillus thuringiensis var. israelensis has been determined by analysis of a recombinant plasmid from Escherichia coli. The hydropathy plot of the protein shows it to be highly hydrophobic, consistent with a postulated cytolytic mechanism of action for the toxin. In addition, the delta-endotoxin transcriptional start points that are used in B. thuringiensis and an E. coli recombinant have been determined. In B. thuringiensis var. israelensis, transcription initiates from a single start point, and gene-specific transcripts are not observed before stage II of sporulation. This is the stage at which delta-endotoxin antigen is first detected, indicating that control of expression is primarily at the transcriptional level for this protein. Analysis of gene-specific transcription in E. coli indicates that at least three start points are utilized in this organism. Interestingly, the highest level of delta-endotoxin mRNA is seen during mid-exponential growth of E. coli and the level appears to decrease as the cells enter the stationary phase of growth.

Amino Acid Sequence↗
PubMed1986

Bacillus thuringiensis var. israelensis delta-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis.

A plasmid-borne gene from Bacillus thuringiensis var. israelensis encoding a 27,340 Mr insecticidal delta-endotoxin has been cloned on a bifunctional multicopy plasmid in a wild-type sporogenic strain and two asporogenic mutants of Bacillus subtilis. The delta-endotoxin gene is expressed at a low level during vegetative growth in all three strains, but the synthesis of the toxin increases markedly during the third hour of stationary phase for both the sporogenic strain and an asporogenic mutant containing the OJ lesion. However, in a stage OA mutant, this increase in delta-endotoxin synthesis is not observed. In both the wild-type sporogenic B. subtilis and the asporogenic OJ strain, phase-bright inclusions, resembling the israelensis crystal in appearance, are visible during late stationary phase. The insoluble inclusions from the B. subtilis transformants, consisting solely of the 27,340 Mr polypeptide, were purified by density gradient centrifugation and found to be extremely toxic to Aedes aegypti larvae. After solubilization in alkaline buffer, this polypeptide was also shown to be haemolytic for human erythrocytes and to lyse Aedes albopictus cells with the same LC50 value as native israelensis delta-endotoxin crystals. During stationary phase, novel mRNA species appear in both the wild-type strain and the OJ mutant, but not in the OA mutant, and these appear to be the major gene-specific transcripts. Transcriptional mapping of delta-endotoxin-specific mRNA has shown that the same region of initiation is used at a relatively low level in all three strains during vegetative growth.

Bacillus subtilis↗