The mutation R(423)S in the Bacillus thuringiensis hybrid toxin CryAAC slightly increases toxicity for Mamestra brassicae L

Bacillus thuringiensis Cry1Ac toxin is 100 times less toxic than Cry1C to Mamestra brassicae. An R(423)S mutation abolishes Cry1Ac toxin proteolysis in M. brassicae gut juice but does not increase its toxicity to this insect. The CryAAC hybrid toxin (1Ac/1Ac/1Ca) is toxic to M. brassicae but is susceptible to gut protease digestion at the R(423) residue. Accordingly we have investigated the effect of the R(423)S mutation in CryAAC on its toxicity for M. brassicae and Pieris brassicae. Bioassays demonstrated that the R(423)S mutation slightly increased the toxicity of CryAAC for M. brassicae by having a significantly inhibitory effect on the growth of surviving larvae. The mutant hybrid was still highly toxic to P. brassicae. Features of CryAACR(423)S such as, (1) stability in M. brassicae gut juice and (2) crystal solubility were investigated. Computer simulations suggest that a possible major increase in flexibility in the CryAAC loop beta7/beta8 (G(391)-P(397)) caused by the R(423)S substitution could be a reason for the increase in M. brassicae toxicity.     

Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity.     

Cloning of ODAP Degrading Gene and Its Expression as Fusion Protein in Escherichia coli

A gene responsible for the degradation of ß-N-Oxalyl diaminopropionic acid (ODAP) was fused to the maIE gene, which codes for maltose binding protein, by cloning into an expression vector pMAL c2. The gene has been expressed as fusion protein of mol wt approximately 62 kD. It has been purified by affinity chromatography. The fusion protein has been cleaved by an endoprotease factor Xa and the presence of maltose binding protein and the product of the cloned gene confirmed. SDS-PAGE has shown that the product of the ODAP degrading gene is a single polypeptide of mol wt of about 20.7 kD.     

Immunological study of cellular components of Pseudomonas aeruginosa. IV. Fractionation of Pseudomonas aeruginosa mucus by diafiltration and biological properties of isolated fractions]

In fractionation of Pseudomonas aeruginosa mucus (strains No. 8 and 1463) by means of diafiltration on the system of membranes Diaflo XM-300, XM-100A, PM-30, and PM-10 there was obtained a successive series of fractions differing by the molecular weight and chemical composition. According to the results of gel chromatography fractions with the mol wt of 100000 dalton and over apparently represented protein-polysaccharide components of mucus in the form of complexes; fractions with the mol wt of 30000 dalton and lower contained a considerable amount of free protein along with the protein-polysaccharide complex. The fractions obtained differed by biological properties: fractions with the mol wt of 100000 dalton and over were toxic for mice and possessed weak antigenic properties in the precipitation in agar test and immunoelectrophoresis; fractions with the mol wt lower than 30000 dalton expressed in the mentioned test distinct antigenic properties and proved to be practically nontoxic for mice. Thus the use of diafiltration method permitted to separate the antigenic, weakly toxic component of Ps. aeruginosa mucus from the toxic factor with weak antigenic properties.     

Characterization of the delta-endotoxin of a Bacillus thuringiensis Isolate Active Against Tsetse, Glossina morsitans, and a Stem Borer, Chilo partellus

The delta-endotoxin crystals of a Bacillus thuringiensis isolate active against the tsetse fly, Glossina morsitans, were isolated from a nutrient broth culture by low speed centrifugation. Analysis of these crystals by denaturing gel electrophoresis revealed that the major component of the crystal delta-endotoxin was a protein of mol. wt ~ 120000. Upon solubilization under alkaline pH and reducing conditions, the crystal yielded a toxin of mol. wt ~ 64 000. Treatment of the toxin with bovine trypsin resulted in a shift in the mol. wt to a toxin of ~ 62000, while treatment with bovine chymotrypsin gave a toxin of ~ 60 000. Methyl green staining revealed that the endotoxin was phosphorylated, while staining with periodic acid schiff reagent showed that it was glycosylated. The carbohydrate moiety was of the high mannose type as shown by staining with fluorescein isothiocyanate conjugated to concanavalin A. Following gel permeation chromatography on a Superose 12 column, the solubilized toxin resolved into six main protein peaks, two of which had trypsin-like activity. The delta-endotoxin caused mortalities in the tsetse, G. morsitans morsitans (LC50 of 42.4mug ml-1) and 4th instar Chilo partellus larvae (LC50 of 53.8 mug ml-1), but had no effect on 3rd instar Aedes aegypti larvae.     

The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them.

The killer character of the yeast Kluyveromyces lactis is associated with the presence of the linear DNA plasmids k1 and k2 and results from the secretion of a protein toxin into the growth medium. We find that toxin activity co-purifies with three polypeptides which we have termed the alpha- (mol. wt 99,000), beta- (mol. wt 30,000) and gamma- (mol. wt 27,500) subunits. The alpha-subunit appears to contain a single asparagine-linked oligosaccharide chain but neither of the smaller subunits is glycosylated. The N-terminal amino acid sequence of each subunit has been determined. Comparison of these data with the DNA sequence of plasmid k1 indicates that it encodes all three subunits. The alpha- and beta-subunits must be processed from the primary translation product of a single gene by an enzyme related to the KEX2 endopeptidase of Saccharomyces cerevisiae.     

Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.     

Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.     

Protein kinase activities associated with distinct antigenic forms of polyoma virus middle T-antigen

The tyrosine-specific protein kinase activity previously described in T-antigens of polyoma virus immunoprecipitated with anti-tumour sera has been investigated using monoclonal antibodies. This activity is associated with middle T-antigen but it can be separated by selective antibody precipitation from the majority of this protein. The difference between active and inactive forms can be accounted for by an antigenic difference at the N terminus of middle T-antigen molecules. Moreover, the two different mol. wt. forms of middle T-antigen that can act as phosphoacceptors have been separated by antibody precipitation and therefore shown to be immunologically distinct. The binding position of the antibody used for immunoprecipitation has been observed to have a quantitative influence on the in vitro protein kinase reaction, in one case appearing to stimulate the activity. The detection of the in vitro protein kinase activity in immunoprecipitates obtained with several different monoclonal antibodies directed against the middle T-antigen indicates that the activity is a property tightly associated with this polyoma virus-coded protein.     

Functional and expression pattern analysis of chemosensory proteins expressed in antennae and pheromonal gland of Mamestra brassicae

Sequences coding for chemosensory proteins (CSP) CSPMbraA and CSPMbraB, soluble proteins of low mol. wt, have been amplified using polymerase chain reaction on antennal and pheromonal gland complementary DNAs. On the basis of their sequences, these proteins could be classed in the 'OS-D like' protein family whose first member was described in Drosophila, and that includes proteins characterized in chemosensory organs of many insect phylla, including our recent identification in Mamestra brassicae proboscis. Binding assays have shown that these proteins bind the pheromonal component (Z)-11-hexadecenyl-1-acetate (Z11-16:Ac) as well as (Z)-11-octadecenyl-1-acetate (Z11-18:Ac), an other putative component of the M. brassicae pheromonal blend. Furthermore, binding with fatty acids, but not with progesterone that is a structurally unrelated compound, leads to the hypothesis that the odorant-binding capability of the MbraCSPs may be restricted to fatty acids and/or to 16-18 carbon backbone skeletons. Thus, these proteins do not show the same highly binding specificity as the pheromone-binding proteins do. The CSP-related proteins appear homologous based on sequence identity, conserved cysteine residues and general patterns of expression. However, phylogenetic analyses suggest the presence of multiple classes of CSP within a given species and possible diversification of CSPs within different orders. This diversity perhaps contributes to the many CSP functions proposed in the literature. In M. brassicae, we localized the CSPMbraA expression to the sensilla trichodea, devoted to pheromone reception, suggesting a role in the chemosensory pathway. However, we also localized such proteins in the pheromonal gland, devoid of any chemosensory structure. This suggests that the M. brassicae CSP could be involved in transport of hydrophobic molecules through different aqueous media, such as the sensillar lymph, as well as the pheromonal gland cytosol.     

Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive.     

Identification of a delta-Endotoxin Gene Product Specifically Active against Spodoptera littoralis Bdv. among Proteolysed Fractions of the Insecticidal Crystals of Bacillus thuringiensis subsp. aizawai 7.29

At least three different insecticidal crystal protein genes were shown to be expressed in Bacillus thuringiensis subsp. aizawai 7.29, a strain that is potentially active against the cotton leafworm Spodoptera littoralis Bdv. Among crude K-60 fractions (60- to 70-kilodalton [kDa] molecules) that were products of proteolysed crystals containing the active domains of the protoxin molecules, we were able to distinguish several distinct components on the basis of their antigenic relationship and their larvicidal properties. A purified fraction designated SF2 was a 61-kDa component specifically active against Pieris brassicae L. and homologous to the B. thuringiensis subsp. berliner 1715 plasmid-encoded crystal protein. A second fraction designated SF1 was composed of 63- and 65-kDa polypeptides and was specifically active against S. littoralis. The SF1 fraction and particularly the 65-kDa component were not antigenically related to the 61-kDa component. The purified fractions were compared with the products of three different crystal protein genes we previously cloned from total DNA of B. thuringiensis subsp. aizawai, among them a new type of crystal protein gene encoding a protein that is specifically active against S. littoralis and other insects of the Noctuidae family. This approach led us to consider the 65-kDa component as a minimum active part of a delta-endotoxin that is encoded by this new gene. Products of the two other cloned genes can be correlated with the 61- and 63-kDa components, respectively. Thus, in B. thuringiensis subsp. aizawai 7.29, multiple delta-endotoxin genes of different structural types direct the synthesis of several delta-endotoxins with different host specificities which were identified as components of the insecticidal crystals.     

Identification of a nuclear antigen with molecular weight of 48,000 differentially expressed in tumour and normal cells

A non-histone protein with mol. wt of 48,000 differentially expressed in normal and tumour cells was identified using immunological criteria. Antibodies were raised against a component specific for Kirkman-Robbins hepatoma of mol. wt about 48,000 separated from hepatoma non-histone proteins by preparative electrophoresis in polyacrylamide gel. It was demonstrated by immunoblotting that Morris hepatoma 7777 and Ehrlich ascites cells share an antigenic non-histone protein with Kirman-Robbins hepatoma. Tumour cells when compared with normal cells, i.e. hamster and rat liver, are characterized by significant enrichment of this component. Intracellular distribution of the polypeptide with mol. wt 48,000 suggests that this component may be a structural protein the biosynthesis of which increases or the antigenic determinants of which change in tumour cells.     

Partial purification of α-glycerotoxin,a presynaptic neurotoxin from the venom glands of the polychaete annelid glycera convoluta

The venom secreted from glands appended to the jaws of Glycera convoluta, a Polychaete Annelid, increases the spontaneous quantal release of transmitter from nerve terminals. The component that is biologically active on vertebrate cholinergic nerve terminals has recently been shown to be a high molecular weight protein. In the present work, the crude extract from the venom apparatus was shown to be toxic for mammals and crustaceans. It was fractionated by gel filtrations and ion exchange chromatographies. The biologically active component at frog neuromuscular junctions, α-glycerotoxin, was purified more than 1,000-fold. It is distinct from the components that are toxic for crustaceans. Purified α-glycerotoxin is a globular protein of 300,000 ± 20,000 mol wt. It has a Stokes radius of 65 Å and a sedimentation coefficient of 11 S. By its molecular properties, α-glycerotoxin appears distinct from other neurotoxins such as α-latrotoxin, which also trigger transmitter release.     

A novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crystal protein

Only one of the four lepidoptera-specific crystal protein subclasses (CryIC) Bacillus thuringiensis was previously shown to be highly toxic against several Spodoptera species. By using a cryIC-derived nucleotide probe, DNA from 25 different strains of B. thuringiensis was screened for the presence of homologous sequences. A putative crystal protein gene, considerably different from the cryIC gene subclass, was identified in the DNA of strain 4F1 (serotype kenyae) and cloned in Escherichia coli. Its nucleotide sequence was determined and appeared to contain several features typical for a crystal protein gene. Furthermore, the region coding for the N-terminal part of the putative toxic fragment showed extensive homology to subclass cryIA sequences derived from gene BtII, whereas the region coding for the C-terminal part appeared to be highly homologous to the cryIC gene BtVI. With an anti-crystal protein antiserum, a polypeptide of the expected size could be demonstrated in Western immunoblots, onto which a lysate of E. coli cells harboring the putative gene, now designated as BtXI, had been transferred. Cells expressing the gene appeared to be equally toxic against larvae of Spodoptera exigua as recombinant cells expressing the BtVI (cryIC)-encoded crystal protein. However, no toxicity against larvae of Heliothis virescens, Mamestra brassicae, or Pieris brassicae could be demonstrated. The nucleotide sequence analysis and the toxicity studies showed that this novel crystal protein gene falls into a new cryl gene subclass. We propose that this subclass be referred to as cryIE.     

Immunological analysis of chemoattractive proteins from earthworm to garter snakes.

1. Two sets of polyclonal antibodies to two highly purified prey-derived snake-attractive proteins, a low molecular weight (3000) protein and a 20,000 mol. wt protein, were generated in rabbits. 2. They are immunospecific for their respective purified immunogens and do not cross-react with each other. 3. Eight prey-derived proteins that elicit attack by garter snakes (Thamnophis sirtalis) from earthworms (Lumbricus terrestris) were analyzed with these antibodies, and can be assigned to three distinct groups on the basis of their antigenic properties. 4. Unfolding or denaturation of the low molecular weight protein did not alter its antigenic activity to its polyclonal antibodies, suggesting the antigenic epitopes contain contiguous amino acid sequences. 5. In contrast, unfolding of the 20,000 mol. wt protein resulted in a loss of its binding with antibodies, suggesting that the epitope of this protein contains noncontiguous amino acid sequences. 6. The snake-attractivity of the 20,000 mol. wt protein could not be neutralized by reacting it with its antiserum, suggesting that the antigenic determinant (the epitope) of the antigen is not an integral part of the attractive domain of the ES20 protein. 7. In contrast, the attractivity of the purified low molecular weight protein could be neutralized by the polyclonal antibodies.     

Turtle-dove ovomucoid, a glycoprotein proteinase inhibitor with P1-blood-group antigen activity.

Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.     

Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac delta-endotoxin

Bacillus thuringiensis protein delta-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac delta-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.     

Diphtheria toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphtheria toxin-related interleukin-2 fusion protein

We have genetically replaced the diphtheria toxin receptor binding domain with a synthetic gene encoding interleukin-2 (IL-2) and a translational stop signal. The diphtheria toxin-related T-cell growth factor fusion gene encodes a 70 586-d polypeptide, pro-IL-2-toxin. The mature form of IL-2-toxin has a deduced mol. wt of 68,086 and is shown to be exported to the periplasmic compartment of Escherichia coli (pABI508), and contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components. IL-2-toxin has been purified from periplasmic extracts of recombinant strains of E. coli (pABI508) by immunoaffinity chromatography using immobilized anti-IL-2. The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL-2 receptor are resistant to IL-2-toxin action.     

A mammal toxin derived from the venom of a chactoid scorpion

1. It has been shown that the low toxicity to mammals (LD50 of about 200 mg per kg mice body weight) of the chactoid scorpion venom Scorpio maurus palmatus (Scorpionidae) is due to a single low molecular weight basic protein. 2. This compound was purified by the aid of gel filtration and ion exchange column chromatography, possessed about 80% of the mice lethality of the crude venom with an increase of about 60 fold in its specific toxicity. 3. It is composed of 32 amino acids (mol. wt = 3478) and devoid of isoleucine, leucine, phenylalanine, histidine and tryptophan. 4. The unique amino acid composition of the present toxin is compared to those of the well known buthoid scorpion venom mammal toxins and some other toxins derived from the same venom. 5. It is the first chemically characterized chactoid toxin.