Activities of Bacillus thuringiensis insecticidal crystal proteins Cyt1Aa and Cyt2Aa against three species of sheep blowfly.

The toxicity of Bacillus thuringiensis Cyt1Aa protein to sheep blowfly larvae depends on its solubilization and proteolytic activation. Cyt1Aa crystals were not toxic. Full-length and trypsin-digested Cyt1Aa proteins were toxic to larvae of three species of sheep blowfly. Neither full-length nor trypsin-digested Cyt2A soluble crystal proteins were toxic.

Identification of an insecticidal crystal protein from Bacillus thuringiensis DSIR517 with significant sequence differences from previously described toxins.

The nucleotide (nt) sequence of a DNA segment containing the majority of a gene cloned from Bacillus thuringiensis DSIR517 encoding a 130 kDa insecticidal crystal protein has been determined. Sequence analysis reveals an open reading frame (ORF) of 3453 nt. The ATG initiation codon, which is preceded by a potential ribosome-binding site sequence, was confirmed by N-terminal amino acid sequencing. The ORF extends beyond the 3' terminus of the cloned fragment; however, the high degree of homology between the deduced amino acid sequence of this ORF and other Cry proteins suggests the clone lacks only five C-terminal amino acids. Making this assumption, the ORF of 3468 nt encodes a protein of 1156 amino acids with an estimated molecular mass of 129700 Da. Analysis of the deduced amino acid sequence reveals a number of features characteristic of Cry proteins. Alignment of the Cry 517 protein sequence with other Cry proteins suggests it is most closely related to the cryIA-E genes but sufficiently different to form a new cryI gene subclass.

Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins.

In the course of sporulation, Bacillus sphaericus produces an inclusion body which is toxic to a variety of mosquito larvae. In this review we discuss the general biology of this species and concentrate on the genetics and physiology of toxin production and its processing in the midgut of the larval host. The larvicide of B. sphaericus is unique in that it consists of two proteins of 51 and 42 kDa, both of which are required for toxicity to mosquito larvae. There is a low level of sequence similarity between these two proteins, which differ in their sequences from all the other known insecticidal proteins of Bacillus thuringiensis. Within the midgut the 51- and 42-kDa proteins are processed to proteins of 43 and 39 kDa, respectively. The conversion of the 42-kDa protein to a 39-kDa protein results in a major increase in toxicity; the significance of the processing of the 51-kDa protein is not known. In contrast to the results with mosquito larvae, the 39-kDa protein is alone toxic for mosquito-derived tissue culture-grown cells, and this toxicity is not affected by the 51-kDa protein or its derivative, the 43-kDa protein. Comparisons of larvae from species which differ in their susceptibility to the B. sphaericus toxin indicate that the probable difference resides in the nature of the target sites of the epithelial midgut cells and not in uptake or processing of the toxin. A similar conclusion is derived from experiments involving tissue culture-grown cells from mosquito species which differ in their susceptibility to the B. sphaericus toxin.

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.

The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity.

After site-directed mutagenesis, the genes coding for the 42- and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the Bacillus subtilis vector pUE (a derivative of pUB18). The levels of expression of the gene products in B. subtilis DB104 and B. sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots. The results indicated that a higher amount of protein was produced in B. subtilis DB104. Electron microscopic examination of B. subtilis DB104 and B. sphaericus 718 containing the 42- and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B. sphaericus 2362 were produced in the latter. Strains producing only the 42- or the 51-kDa protein were not toxic to larvae of C. pipiens. A mixture of both strains, a single strain producing both proteins, or a fusion of the 51- and the 42-kDa proteins was toxic. The amount of B. subtilis DB104 containing the 42- and the 51-kDa proteins necessary to kill 50% of the larvae of C. pipiens was 5.6 ng (dry weight) of cells per ml. This value was significantly lower than that for B. sphaericus 2362 (14 ng [dry weight] per ml). Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein this protein to primarily 39- and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa. Past studies involving purified proteins from B. sphaericus 2362 indicate an associate of toxicity with the 39-kDa peptide. The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.

Sequence analysis of the mosquitocidal toxin genes encoding 51.4- and 41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297.

The nucleotide sequences of a 3,479-base-pair HindIII DNA fragment from Bacillus sphaericus 2362 and a 2,940-base-pair fragment from strain 2297 were determined; only minor differences were detected between them. Each contained two open reading frames coding for proteins of 51.4 and 41.9 kilodaltons. Both proteins were required for toxicity to larvae of the mosquito Culex pipiens.

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.

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin.

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Sporulation-associated activation of Bacillus sphaericus larvicide.

Preparations of the larvicidal crystal from 46-h cultures of Bacillus sphaericus 2362 contain 125-, 110-, 63-, and 43-kilodalton (kDa) proteins (P. Baumann, B. M. Unterman, L. Baumann, A.H. Broadwell, S.J. Abbene, and R.D. Bowditch, J. Bacteriol. 163:738-747, 1985). The 63- and 43-kDa proteins, which have been purified, are not immunologically cross-reactive, and only the 43-kDa protein is toxic to mosquito larvae. Since antigenic determinants of the two smaller proteins have been detected in the higher-molecular-weight proteins (125 and 110 kDa), it has been suggested that the latter are precursors of the 43- and 63-kDa peptides. In the present study, purified 110-kDa protein was found to be toxic to the larvae of Culex pipiens (50% lethal concentration = 115 ng/ml). A luciferase-luciferin assay for intracellular ATP as well as an assay based on the exclusion of Trypan Blue by live cells indicated that the 110-kDa protein had no effect on tissue-culture-grown cells of C. quinquefasciatus, while cells exposed to the 43-kDa protein rapidly lost viability (50% lethal concentration = 54 microgram(s)/ml by the intracellular ATP assay). These findings suggested that the 110-kDa protein and, by extension, the 125-kDa protein are protoxins which are activated during sporulation by cleavage to a 43-kDa toxin. To further investigate the origins and relationships of the crystal proteins of B. sphaericus, we analyzed samples during the growth and sporulation of the culture. Synthesis of crystal proteins was initiated at the end of exponential growth and was completed after about 7 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high-molecular-weight precursors.

Crystals were purified from spore-crystal complexes of Bacillus sphaericus 2362 by disruption in a French pressure cell followed by centrifugation through 48% (wt/vol) NaBr. Crystals from such preparations had a 50% lethal concentration of 6 ng of protein per ml for the larvae of the mosquito Culex pipiens. When subjected to polyacrylamide gel electrophoresis under denaturing conditions, the proteins in B. sphaericus crystals migrated in positions corresponding to 43, 63, 98, 110, and 125 kilodaltons (kDa); solubilization of the crystal at pH 12 with NaOH eliminated all but the bands at 43 and 63 kDa. Since NaOH-solubilized preparations were toxic to mosquito larvae, these proteins were purified to electrophoretic homogeneity and antiserum was obtained to each. Analysis of the two purified proteins indicated that the 43-kDa protein was toxic to mosquito larvae (50% lethal concentration, 35 ng of protein per ml), whereas the 63-kDa protein was not. Further differences between them were their amino acid compositions, their lack of immunological cross-reactivity, their opposite net charges at pH 7.5, and their susceptibility to digestion by larval midgut proteases (the 63-kDa protein was highly susceptible, whereas the 43-kDa protein was not). The sequence of the 40 N-terminal residues of the 43-kDa protein was determined and found to contain a high percentage of hydrophobic amino acids. The sequence of the 63-kDa protein could not be determined, since it had multiple N termini. By electrophoretically separating the crystal proteins and then electroblotting onto nitrocellulose paper and visualizing the bands with antisera to the 43- and 63-kDa proteins in conjunction with an immunoblot assay, it was found that the high-molecular-mass crystal proteins (98 to 125 kDa) contained antigenic determinants of both proteins. These results suggested that the lower-molecular-weight crystal proteins detected in polyacrylamide gels after electrophoresis under denaturing conditions were derivatives of one or more of the higher-molecular-weight crystal proteins. In vivo studies of the products of crystal degradation by larvae of Culex pipiens indicated that the high-molecular-weight proteins and the 63-kDa antigenic determinants were rapidly degraded and that a 40-kDa protein related to the 43-kDa toxin persisted for the duration of the experiment (4 h). Some of the studies performed with B.sphaericus 2362 were extended to strains 1593, 1691, and 2297 of this species with results which indicated a high degree of similarity between the crystal proteins of all these larvicidal strains.

A comparison of antimicrobial activity of four disclosant dyes.

Four disclosant dyes were tested for antimicrobial activity against 27 oral reference strains and five non-oral human pathogens. Erythrosine and fluorescein inhibited most gram-positive and gram-negative organisms associated with dental plaque, and were bactericidal to selected strains. Fast green and brilliant blue demonstrated little antimicrobial activity.