Identification of the peptides of the crystals of Bacillus thuringiensis var israelensis involved in the mosquito larvicidal activity

Tryptic digestion of the proteins from the purified crystals of B.thuringiensis var israelensis resulted in the decline of high molecular weight peptides without the loss of mosquito larvicidal activity, measured after immobilization of the digests with DEAE- Sephadex A 50 beads. Amongst the peptides generated (less than 44 kDa), a 21 kDa peptide was immunoreactive to the crystal antiserum. Analysis of the peptides released from spores of the toxic (Cry+) and non-toxic (Cry-) strains has revealed a pattern in which only the 26kDa peptide was missing in the Cry-strain. Sporulation and crystal formation were dissociated by the addition of the antibiotic netropsin, which could also inhibit the crystal assembly, without considerable decrease of the larvicidal activity and retention of the 26kDa peptide. These results implicate the 26kDa peptide in the larvicidal action.     

Effect of removal of the cytolytic factor of Bacillus thuringiensis subsp. israelensis on mosquito toxicity

Solubilized crystal protein of Bacillus thuringiensis subsp. israelensis was fractionated by affinity chromatography using a monoclonal antibody directed against the crystal's 28 kDa peptide. The 28 kDa peptide was found to be relatively nontoxic to mosquito larvae although it does contain the hemolytic activity of the crystals. The crystal protein fraction depleted of the 28 kDa peptide was found to be nonhemolytic and to retain nearly full toxicity to mosquito larvae. These results suggest that the 28 kDa peptide is not required for the toxicity of solubilized crystal protein.     

Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae.

A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described. The crystal proteins are produced as bipyramidal crystals. These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa. Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested. In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae. The crystal protein-encoding gene was cloned and sequenced. The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively. Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins. Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins. CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae. Natural CryIIIC crystals are not toxic. Toxicity is revealed only after an in vitro solubilization and activation step.     

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Summary A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.     

Enzymatic activity and inhibition of the neurotoxic complex vipoxin from the venom of Vipera ammodytes meridionalis

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides.     

Toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa contain sequences homologous to peptide synthetase genes

Abstract Toxic strains of Microcystis aeruginosa produce cyclic heptatoxins (microcystins) that are believed to be synthesized non-ribosomally by peptide synthetases. We analysed toxin-producing and non-toxic strains of M. aeruginosa with respect to the presence of DNA sequences potentially encoding peptide synthetases. Hybridizations of genomic DNA of various M. aeruginosa strains with PCR-amplificated fragments possessing homologies to adenylate-forming domains of peptide synthetase genes provided first evidence for the existence of corresponding genes in cyanobacteria. Furthermore we isolated and sequenced from genomic libraries overlapping fragments of M. aeruginosa DNA with a total length of 2982 bp showing significant homology to genes encoding peptide synthetases and hybridizing exclusively with DNA from toxic strains. Our results indicate that both toxic and non-toxic strains of M. aeruginosa possess genes coding for peptide synthetases and that hepatotoxin-producing and non-toxic strains differ in their content of genes for specific peptide synthetases.     

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.     

Identification and purification of GTP-binding regulatory proteins from plasma membranes of swine heart

A 23 kDa GTP-binding protein was purified from pig heart sarcolemma. This protein was not ADP-ribosylated by cholera, pertussis and botulinum C3 toxins. In pig heart sarcolemma pertussis toxin ADP-ribosylated 40 kDa subunit of Gi-protein, cholera toxin--45 kDa subunit of Gs-protein, botulinum C3 toxin ADP-ribosylated a group of proteins with Mr 22, 26 and 29 kDa. Antiserum generated against the peptide common for all alpha-subunits of G-proteins did not react with purified 23 kDa protein. Trypsin cleaved the 23 kDa protein in the presence of guanyl nucleotides into a 22 kDa fragment. Proteolysis of the 39 kDa alpha 0-subunit from bovine brain plasma membranes and ADP-ribosylated 40 kDa alpha i-subunit from pig heart sarcolemma in the presence of GTP gamma S yielded the 37 and 38 kDa fragments, respectively. In the presence of GTP and GDP the proteolysis of alpha 0 yielded the 24 and 15 kDa fragments, while the proteolysis of ADP-ribosylated alpha i-subunit yielded a labelled 16 kDa peptide. Irrespective of nucleotides trypsin cleaved the ADP-ribosylated 26 kDa substrate of botulinum C3 toxin into two labelled peptides with Mr 24 and 17 kDa. The data obtained indicate the existence in pig heart sarcolemma of a new 23 kDa GTP-binding protein with partial homology to the alpha-subunits of "classical" G-proteins.     

Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina

Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.     

Characterization of INTA 51-3, a New Atypical Strain of Bacillus thuringiensis from Argentina

Several isolates of Bacillus thuringiensis native to Argentina obtained in a nationwide screening program showed atypical crystal morphology. One of these strains, INTA 51-3, was further characterized in order to determine other features like protein composition of its parasporal crystal, plasmid pattern, identification of cry genes and toxicological properties. B. thuringiensis INTA 51-3 (serovar tohokuensis) had an amorphous inclusion containing a major protein component of ca. 130 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique protease-resistant peptide of ca. 90 kDa. The plasmid pattern from INTA 51-3 resembled that of the standard strain HD-1. However, Southern analysis showed no hybridization to fragments of cry1Aa, cry2Aa, cry3A, and cry11A genes. Degenerate primers were used for identification of the cry1 genes by PCR. Nevertheless, the presence of cry1 type gene(s) in B. thuringiensis INTA 51-3 was confirmed. Highly concentrated crystal suspensions showed to be weakly toxic only to lepidopteran species. Received: 23 May 2000 / Accepted: 26 June 2000     

Identification of a novel light-harvesting complex II protein (LHC IIc′)

The caroteno-chlorophyll-protein, LHC IIc, is a relatively minor component of the PS II antenna. Isolated LHC IIc contains a major protein of 28 kDa along with a 26 kDa subunit in lower abundance. Previously, it was not known if the 26 kDa protein was closely related to the 28 kDa LHC IIc protein or if it was a comigrating LHC IIb contaminating subunit. A sequence of 20 amino acid residues was obtained by direct protein micro-sequencing of an internal cyanogen bromide-derived peptide fragment of the 26 kDa protein isolated from barley. The sequence shows, and antibody reactions confirm, that the 26 kDa protein is similar but distinct from both the 28 kDa LHC IIc and LHC IIb protein sequences, indicating that there remains at least one more cab gene to be identified in higher plants. Furthermore, it is difficult to interpret the data in any way other than that there is a novel LHC II pigment-protein (LHC IIc) that co-migrates with LHC IIc.Abbreviations CC core complex - LHC light-harvesting complex - PVDF polyvinylidene fluoride     

Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate.

We have used native gel electrophoresis followed by fluorogenic peptide overlay to identify multiple forms of rabbit reticulocyte multicatalytic protease (MCP) or 20 S protease, and two forms of rabbit 26 S ubiquitin/ATP-dependent protease. An abundant, fast-migrating 20 S complex (20 SF) possesses modest ability to hydrolyze the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide. In contrast, two minor, slower migrating species cleave the peptide at high rates. A unique 30-kDa polypeptide is associated with one of the active MCPs, and a 160-kDa subunit is associated with the other. Two electrophoretically distinct 26 S proteases can also be isolated from rabbit reticulocyte lysate. The faster migrating form, 26 SF, is more resistant to inactivation by ATP depletion. Despite the differential response to nucleotides and the distinctive electrophoretic mobilities of 26 SF and 26 SS, we have not identified any subunit differences between the two enzymes. In addition to active 26 S proteases, we have discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26 S protease (e.g. molecular masses between 30 and 110 kDa). Incubation of this protein complex with purified MCP and ATP results in the formation of the 26 S proteases.     

Selenium-containing proteins of rat kidney and liver microsomes

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.     

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.     

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.     

Role of tryptophan 26 in the NAD glycohydrolase reaction of the S-1 subunit of pertussis toxin

The gene encoding a catalytically active deletion peptide, the C180 peptide, of the S-1 subunit of pertussis toxin was engineered to facilitate mutagenesis at the Trp-26 (wild-type) coding sequence. A synthetic double-stranded oligonucleotide was inserted into the C180 gene such that all possible codons would be introduced into position 26. Seven individual mutants of the C180 peptide which possessed amino acid substitutions at residue 26 (collectively termed C180W26n peptides) were purified from periplasmic extracts of Escherichia coli. Each C180W26n peptide was present as a single major peptide that had an apparent molecular mass of between 20.9 and 24.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and each showed similar immunoreactivity relative to the C180 peptide. The C180W26n peptides demonstrated marked reduction of both ADP-ribosyltransferase and NAD glycohydrolase activities at 25 nM and 10 microM NAD, respectively. Kinetic analysis of the two most active mutants, C180W26F and C180W26Y, revealed that the major perturbation of NAD glycohydrolase activity was due to an increase (approximately 20-fold) in the Km for NAD between these mutants and the C180 peptide.     

Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences.

During sporulation, Bacillus sphaericus 2362 produces a parasporal crystalline protein which is toxic for the larvae of a number of mosquito species. Using the Escherichia coli cloning vector lambda gt11, in which gene products of the inserts may be fused to beta-galactosidase, we isolated 29 bacteriophages which produced peptides-reacting with antiserum to crystal protein. On the basis of restriction enzyme analyses of the recombinants and Ouchterlony immunodiffusion experiments with induced lysogens as a source of antigens, the recombinants were assigned to three groups, designated A, B, and C. Group A consisted of three clones which appeared to express all or part of the B. sphaericus toxin gene from their own promoters and one clone producing a beta-galactosidase-toxin fusion protein. The host cells of two induced recombinant lysogens of this group were toxic to larvae of Culex pipiens. A cell suspension containing 174 ng (dry weight) of the more toxic recombinant per ml killed 50% of the larvae. Both recombinants formed peptides with molecular sizes of 27, 43, and 63 kilodaltons (kDa). The antigenically related 27- and 43-kDa peptides were distinct from the 63-kDa peptide, which resembled crystals from sporulating cells of B. sphaericus in which antigenically distinct 43- and 63-kDa proteins are derived from a 125-kDa precursor. A 3.5-kilobase HindIII fragment from recombinants having toxic activity against larvae was subcloned into pGEM-3-blue. E. coli cells harboring this fragment were toxic to mosquito larvae and produced peptides of 27, 43, and 63 kDa. The distribution of the A gene among strains of B. sphaericus of different toxicities suggested that it is the sole or principal gene encoding the larvicidal crystal protein. The two recombinants of group B and the 23 of group C were all beta-galactosidase fusion proteins, suggesting that in E. coli these genes were not readily expressed from their own promoters. The distribution of these two genes in different strains of B. sphaericus suggested that they do not have a role in the toxicity of this species to mosquito larvae.     

Delineation of a toxin-encoding segment of a Bacillus thuringiensis crystal protein gene

Crystals of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel contain a Mr 134,000 protoxin which can be cleaved by proteolysis to a peptide of Mr approximately 70,000; this peptide is lethal to lepidopteran larvae. We have analyzed the peptides produced by recombinant Escherichia coli strains bearing deletions and fusions of the protoxin gene in order to delineate the portion of the gene which encodes the toxic peptide. The recombinant strains produced the toxic peptide as well as larger peptides whose size was related to the length of the deleted gene. The results indicate that the amino-terminal 55% of the protoxin protein is sufficient for toxicity. While two different gene fusions to the 10th codon allowed the synthesis of toxic polypeptides, fusions to the 50th codon did not. 3' end deletions up to the 645th codon allowed synthesis of the toxic peptide whereas a deletion to the 603rd codon yielded a non-toxic peptide. Some of the 5' and 3' end alterations to the gene caused changes in the proteolytic cleavage patterns of the polypeptides synthesized by E. coli, suggesting that the alterations led to conformational changes in the proteins. The presence of different 3' end segments affected the levels of synthesis of the altered crystal proteins.     

Purification and comparison of the structures of human liver acidic alpha-D-mannosidases A and B.

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.