Functional Complementation of Nontoxic Mutant Binary Toxins of Bacillus sphaericus 1593M Generated by Site-Directed Mutagenesis

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.     

Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin.

Bacillus sphaericus 2362 produces a binary toxin consisting of 51- and 42-kDa proteins, both of which are required for toxicity to mosquito larvae. Upon ingestion by larvae, these proteins are processed to 43 and 39 kDa, respectively. Using site-directed mutagenesis, we have obtained N- and C-terminal deletions of the 51-kDa protein and expressed them in B. subtilis by using the subtilisin promoter. Removal of 21 amino acids from the N terminus and 53 amino acids from the C terminus resulted in a protein with the same electrophoretic properties as the 43-kDa degradation product which accumulates in the guts of mosquito larvae. This protein was toxic only in the presence of the 42-kDa protein. A deletion of 32 amino acids at the N terminus combined with a 53-amino-acid deletion at the C terminus resulted in a protein which retained toxicity. Toxicity was lost upon a further deletion of amino acids at potential chymotrypsin sites (41 at the N terminus, 61 at the C terminus). Comparison of the processing of the 51- and the 42-kDa proteins indicated that in spite of their sequence similarity proteolysis occurred at different sites.     

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush-border membranes from mosquito larvae.

The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from Culex pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Direct binding and homologous competition experiments indicated a single class of B. sphaericus toxin receptors, with a dissociation constant (Kd) of approximately 20 nM and a maximum binding capacity (Bmax) of approximately 7 pmol/mg BBMF protein. The sugars GalNAc, GlcNAc and N-acetyl neuraminic acid had no detectable inhibitory effect on toxin binding to C. pipiens BBMF. Binding experiments with the non-susceptible mosquito species Aedes aegypti failed to detect significant binding of B. sphaericus binary toxin to A. aegypti BBMF.     

Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean

A 43-kDa soybean protein is a receptor-like protein kinase that is capable of interaction with a 4-kDa hormone-like peptide (leginsulin). The 43-kDa protein consists of alpha and beta subunits; the beta subunit has protein kinase activity that is stimulated by the binding of the 4-kDa peptide. The protein kinase activity is believed to be an early step in a signal transduction cascade, triggered by the peptide. Animal insulin also interacts with the 43-kDa protein and stimulates the protein kinase activity, suggesting that the 4-kDa peptide and insulin bind to the 43-kDa protein with similar mechanisms. To determine the mechanism of interaction between the 4-kDa peptide and 43-kDa protein, we investigated the binding region of the 4-kDa peptide on the 43-kDa protein using surface plasmon resonance (SPR) spectroscopy. We found that the N- (amino acids 1-43) and C-terminal (amino acids 228-251) regions of the alpha subunit of the 43-kDa protein are involved in the binding. The interactions of both insulin and the 4-kDa peptide with the 43-kDa protein were compared using SPR spectroscopy, revealing that insulin binds to the C-terminal regions of the alpha subunit of the 43-kDa protein. These results suggest that the C-terminal region is especially important for the biological function. The N-terminal region is thought to play an important role in stabilizing the complex of the 43-kDa protein and the 4-kDa peptide.     

Modification of the Bacillus sphaericus 51- and 42-kilodalton mosquitocidal proteins: effects of internal deletions, duplications, and formation of hybrid proteins.

The 51- and 42-kDa proteins which constitute the binary mosquitocidal toxin of Bacillus sphaericus 2362 have a low overall sequence similarity but share several regions of near identity (L. Baumann, A. H. Broadwell, and P. Baumann, J. Bacteriol. 170:2045-2050, 1988). By using site-directed mutagenesis, deletions of 6 to 16 amino acids in three of these regions of the 51- and 42-kDa proteins were made, and the modified proteins were expressed in Bacillus subtilis. Deletions in both of these proteins resulted in a loss of toxicity for mosquito larvae. Hybrid proteins containing exchanged fragments of the 51- and 42-kDa proteins were inactive when tested in a variety of combinations, thereby indicating that potentially analogous fragments of these two proteins were not functionally equivalent. An internal duplication of 73 amino acids in the 51-kDa protein and 72 amino acids in the 42-kDa protein resulted in a major reduction in toxicity. These results indicate that the conserved regions of the 51- and 42-kDa proteins are necessary for toxicity to larvae and that the 51- and 42-kDa proteins, despite their sequence similarity, are unique, differing from each other by at least one essential attribute.     

Modification of the Bacillus sphaericus 51- and 42-kilodalton mosquitocidal proteins: effects of internal deletions, duplications, and formation of hybrid proteins

The 51- and 42-kDa proteins which constitute the binary mosquitocidal toxin of Bacillus sphaericus 2362 have a low overall sequence similarity but share several regions of near identity (L. Baumann, A. H. Broadwell, and P. Baumann, J. Bacteriol. 170:2045-2050, 1988). By using site-directed mutagenesis, deletions of 6 to 16 amino acids in three of these regions of the 51- and 42-kDa proteins were made, and the modified proteins were expressed in Bacillus subtilis. Deletions in both of these proteins resulted in a loss of toxicity for mosquito larvae. Hybrid proteins containing exchanged fragments of the 51- and 42-kDa proteins were inactive when tested in a variety of combinations, thereby indicating that potentially analogous fragments of these two proteins were not functionally equivalent. An internal duplication of 73 amino acids in the 51-kDa protein and 72 amino acids in the 42-kDa protein resulted in a major reduction in toxicity. These results indicate that the conserved regions of the 51- and 42-kDa proteins are necessary for toxicity to larvae and that the 51- and 42-kDa proteins, despite their sequence similarity, are unique, differing from each other by at least one essential attribute.     

Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides.

Clones expressing regions of the 100-kDa Bacillus sphaericus SSII-1 mosquitocidal toxin (Mtx) as fusion proteins with glutathione S-transferase were constructed, and the toxin-derived peptides were purified. The in vitro ADP-ribosylation activities of these peptides and their effects on larvae and cells in culture were studied. Mtx25 (amino acids 30 to 493) was found to ADP-ribosylate two proteins with molecular masses of 38 and 42 kDa, respectively, in Culex quinquefasciatus (G7) cell extracts, in addition to ADP-ribosylating itself. Mtx21 (amino acids 30 to 870; or a combination of Mtx25 and Mtx26 (amino acids 259 to 870) caused mortality in C. quinquefasciatus larvae. Mtx25, Mtx26, or Mtx24 (amino acids 30 to 276) alone and Mtx24 in combination with Mtx26 were not toxic to larvae. Mtx21 and Mtx26 produced marked morphological changes in G7 cells and to a lesser extent in Aedes aegypti cells but had no effect on Anopheles gambiae or HeLa cells. Thus, a domain in the N-terminal region of the Mtx protein is sufficient for ADP-ribosylation of C. quinquefasciatus cell protein, and a domain in the C-terminal region is sufficient for toxicity to cultured C. quinquefasciatus cells; however, both regions are necessary for toxicity to mosquito larvae.     

Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1.

The 97-kDa protein Mtx21, derived from the 100-kDa mosquitocidal protein (Mtx) from Bacillus sphaericus SSII-1 by the deletion of the putative signal sequence, was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and the fusion protein was purified by affinity chromatography. The fusion protein bound to glutathione agarose was cleaved with thrombin to release the Mtx21 protein. The 97-kDa Mtx21 protein was found to be toxic to Culex quinquefasciatus larvae with a 50% lethal concentration of 15 ng/ml. Treating Mtx21 with crude mosquito larval gut extracts gave rise to two major peptides of 70 and 27 kDa. Treating the 97-kDa Mtx21 protein with trysin also gave rise to a similar proteolytic cleavage pattern. N-terminal sequencing showed that the 27-kDa peptide was derived from the N-terminal region of the 97-kDa protein and that the 70-kDa protein was from the C-terminal region of the 97-kDa protein. The 27-kDa peptide has all the previously identified regions of homology with the catalytic peptides of the ADP-ribosyltransferase toxins, such as pertussis toxin S1 peptide, while the 70-kDa peptide has three internal regions of homology.     

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

Bacillus sphaericus produces a mosquitocidal binary toxin composed of two subunits, BinA (42 kDa) and BinB (51 kDa). Both components are required for maximum toxicity against mosquito larvae. BinB has been proposed to provide specificity by binding to the epithelial gut cell membrane, while BinA may be responsible for toxicity. To identify regions in BinB responsible for receptor binding and for interaction to BinA, we used six BinB shorter constructs derived from both the N-terminal and the C-terminal halves of the protein. All constructs expressed as inclusion bodies in Escherichia coli, similarly to the wild-type protein. A marked decrease in larvicidal activity was observed when BinA was used in combination with these BinB constructs, used either individually or in pairs from both N and C-halves of BinB. Nevertheless, immunohistochemistry analyses demonstrate that these constructs are able to bind to the epithelium gut cell membrane, and in vitro protein-protein interaction assays revealed that these constructs can bind to BinA. These results show that fragments corresponding to both halves of BinB are able to bind the receptor and to interact with BinA, but both halves are required by the toxin to exhibit full larvicidal activity.     

Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin

The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the alpha4-alpha5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr(178), Gln(180), Asn(183), Asn(185), and Asn(195)) in the hydrophobic helix-alpha5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in alpha5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn(183) located in the middle of the transmembrane alpha5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes.     

Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae).

The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.     

The N-terminal third of the BinB subunit from the Bacillus sphaericus binary toxin is sufficient for its interaction with midgut receptors in Culex quinquefasciatus

Heterodimeric binary (Bin) toxin, the major insecticidal protein from Bacillus sphaericus, acts on Culex quinquefasciatus larvae through specific binding to the midgut receptor Cqm1, a role mediated by its 448-amino-acid-long BinB subunit. The molecular basis for receptor recognition is not well understood and this study attempted to identify protein segments and amino acid motifs within BinB that are required for this event. First, N- and C-terminally truncated constructs were evaluated for their capacity to bind to native Cqm1 through pull-down assays. These showed that residues N33 to L158 of the subunit are required for Cqm1 binding. Nine different full-length mutants were then generated in which selected blocks of three amino acids were replaced by alanines. In new pull-down assays, two mutants, in which residues (85) IRF(87) and (147) FQF(149) were targeted, failed to bind the receptor. Competition binding assays confirmed the requirements for the N-terminal 158 residues, and the (147) FQF(149) epitope, for the mutant proteins to compete with native Bin toxin when binding to membrane fractions from the insect midgut. The data from this work rule out the involvement of C-terminal segments in receptor binding, highlighting the need for multiple elements within the protein's N-terminal third for it to occur.     

Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).     

Construction by site-directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva-processed toxin of Bacillus sphaericus 2362.

After ingestion of the parasporal crystals of Bacillus sphaericus, mosquito larvae process the 42-kilodalton (kDa) toxin to a protein of 39 kDa, which has an increased toxicity (A. H. Broadwell and P. Baumann, Appl. Environ. Microbiol. 53:1333-1337, 1987). A similar activation is performed by trypsin and chymotrypsin. Using site-directed mutagenesis, we have constructed derivatives of the 42-kDa toxin with a deletion of 10 amino acids at the N terminus and deletions of 7, 17, or 20 amino acids at the C terminus. Toxicity for mosquito larvae was retained upon deletion of 7 or 17 amino acids but was lost upon deletion of 20 amino acids. Evidence is presented indicating that the protein containing deletions of 10 amino acids at the N terminus and 17 amino acids at the C terminus (corresponding to potential chymotrypsin cleavage sites) is similar to the 39-kDa protein produced in mosquito larvae or by digestion with chymotrypsin. Digestion with trypsin appears to generate a protein lacking 16 or 19 amino acids from the N terminus and 7 amino acids from the C terminus. As is the case with the recombinant-made 42-kDa protein, toxicity of its derivatives is dependent on the presence of a 51-kDa protein which is a component of the parasporal crystal of B. sphaericus 2362.     

Genetic determinants of host ranges of Bacillus sphaericus mosquito larvicidal toxins.

The 51.4-kDa-41.9-kDa binary toxin produced by different strains of Bacillus sphaericus shows differential activity toward Culex quinquefasciatus, Aedes atropalpus, and Aedes aegypti mosquito larvae. The patterns of larvicidal activity toward all three mosquito species and growth retardation in A. aegypti have been shown to be due to the 41.9-kDa protein. By using mutant toxins expressed in Escherichia coli, insecticidal activity and growth retardation correlated with amino acids centered around position 100 of the 41.9-kDa protein. In its response to these toxins, A. atropalpus resembled C. quinquefasciatus rather than its congener, A. aegypti.     

Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity

The two genes of Bacillus sphaericus 1953M coding for the 51.4-kDa and 41.9-kDa proteins are both required for the expression of the active larvicidal toxin in Escherichia coli. The minimal size of the active peptide of the 41.9-kDa toxin was defined by in vitro deletion analysis of the gene and found to consist of 338 amino acids (38.3 kDa). N-terminal deletions past the Ile18 residue and C-terminal deletions past the His352 residue result in the loss of toxic activity and rapid degradation of such modified toxins by host proteases. The minimal active 38.3-kDa peptide produced in E. coli seems to mimick the stable processed form of the toxin found in larval midguts. However, it still requires the action of the synergistic 51.4-kDa protein for the larvicidal activity.     

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]     

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.     

Expression of mosquitocidal toxin genes in a gas-vacuolated strain of Ancylobacter aquaticus.

A series of plasmids bearing the binary toxin genes of Bacillus sphaericus 2297 or 2317.3, the 100-kDa toxin gene of B. sphaericus SSII-1, or the 130-kDa (cryIVB) toxin gene of Bacillus thuringiensis subsp. israelensis were constructed and introduced into Ancylobacter aquaticus by electroporation. The transformed A. aquaticus cells exhibited significant toxicity towards mosquito larvae, demonstrating a potential use of recombinant A. aquaticus for biological control of mosquitoes.     

Tightly bound binary toxin in the cell wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.