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Bacillus thuringiensis subsp, israelensis produces a potent mosquitocidal protein, Cry4A. We have identified a 15-bp catabolite responsive element (cre), overlapping the -35 element of the cry4A promoter. Changing a guanine to adenine at position -49 in the promoter abolished glucose catabolite repression of cry4A and enhanced promoter activity two- to threefold. This cis regulatory element is essential for controlled toxin synthesis, vital to evolutionary success of B. thuringiensis subsp, israelensis.

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation that are extensively used in the field for control of mosquito populations. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in resistant colony of culex quinquefasclatus, apparently due to the production of Cry11A but not Cry4A or Cry4B.

[beta]-Exotoxin I is an insecticidal nucleotide analogue secreted by various Bacillus thuringiensis strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated sigW-ecfX-ecfY, that is essential for [beta]-exotoxin I production in B. thuringiensis subsp, thuringiensis strain 407-1. In this strain, the disruption of the sigW cluster resulted in nontoxic culture supernatants. sigW encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from Bacillus anthracis strain Ames and Bacillus cereus strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from Clostridium perfringens and SigW and SigX from Bacillus subtilis, respectively. EcfX, encoded by the gene following sigW, significantly repressed the expression of sigW when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable cry plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1([Cry.sup.-])([Pig.sup.+]), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of sigW indicated that this gene was expressed during the stationary phase and only in the 407-1 ([Cry.sup.-])([Pig.sup.+]) mutant. This suggests that in the wild type-407([Cry.sup.+]) strain, [beta]-exotoxin I was produced from determinants located on a cry gene-bearing plasmid and that sigW is able to induce [beta]-exotoxin I production in B. thuringiensis in the absence of cry gene-bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that [beta]-exotoxin I production in B. thuringiensis can be restored or induced via an alternative pathway that requires sigW expression.

The Cry1A toxin of Bacillus thuringiensis interacts with more toxin-binding proteins when it is denatured. These toxin-binding proteins make the insect resistant to the toxin.

Domain III of the Bacillus thuringiensis Cry1Ac toxin maintains the structure of the protein and also participates in the formation of the ion channel inside the midgut of insects. Arginine residues at positions 525, 527, 529, and 531 appear to be important for this purpose.

Researchers describe a vegetative insecticidal protein (VIP) from Bacillus thuringiensis that is toxic to several lepidopteran pests. The amino acid sequence differs slightly from other bacterial VIPs and it may have a different mode of action as well. VIPS are produced during the vegetative growth phase of the bacterium.

The Cry1Ja and Cry1A toxins of Bacillus thuringiensis share binding sites in six moth and butterfly species from different families. This means that the insects might develop cross-resistance to the two toxins.

Residue Asn135 in alpha-helix 4 of the Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins is necessary for pore formation and toxin oligomerization in the gut epithelium of Manduca sexta. However, a mutant protein created by substituting Gln for Asp still binds to the epithelium.

A 20-kDa helper protein in Bacillus thuringiensis enhances the production of Cry1Ac toxin. The P20 protein increases toxin yield more than two-fold, produces larger crystals, and Cry1Ac is more toxic against Heliothis armigera larvae when synthesized in the presence of the helper protein.

The diamondback moth has two genes that confer resistance to the Bacillus thuringiensis Cry toxins. One is dominant and one is partially recessive. Cross-resistance among insects from these two lines also differs.

Differences in packaging of the delta-endotoxins of Bacillus thuringiensis may account for the fact that the Cry1Da1 endotoxin is present in much smaller amounts in inclusions than the other endotoxins. The inclusions are ingested by susceptible insect larva and the protoxins are metabolized to toxins.

Strains of the diamondback moth Plutella xylostella have different sensitivities to the Cry toxins of Bacillus thuringiensis depending on what part of the world they come from. Strains from England, Hawaii, and the Netherlands had different sensitivities but in all three, Cry1Ab and Cry1Ac were the most toxic and Cry1Da the least toxic.

Some insecticidal crystal toxins from Bacillus thuringiensis are less effective in inducing pores in membranes at a high pH. This was the conclusion of researchers who tested the Cry1Ac, Cry1C, and Cry1E toxins.

Researchers show that the P20 protein from Bacillus thuringiensis interacts with the Cry11A protein, making it more toxic to mosquito larvae. This was confirmed in Escherichia coli and Pseudomonas putida that had been genetically engineered to produce both proteins.

Insects possess varying degrees of resistance to the Bacillus thuringiensis Cry1Ab and Cry1Ac toxins. The resistance appears to be mediated by a protease.

Some insects may be resistant to the Cry1Ac delta-endotoxin of Bacillus thuringiensis because they contain intestinal proteases that completely degrade the toxin. Some degree of proteolysis is necessary to activate the toxin inside the insect, but too much can inactivate the toxin.

The 130 kDA delta-endotoxin produced by Bacillus thuringiensis is cleaved into 20 and 45 kDa fragments, which can form a 60 kDa complex. This 60 kDa complex is toxic to insect larvae, whereas the 20 and 45 kDa fragments separately are not.

The Cyt1Aa protein from Bacillus thuringiensis subsp. israelensis, which was previously known to be toxic only to mosquitoes and related dipterans, was observed to be highly toxic to the cottonwood leaf beetle Chrysomela scripta. Also, Cyt1Aa was found to counter high levels of resistance in C. scripta which have resistance to Cry3Aa. Substantial cross-resistance to Cry1Ba in the Cry3Aa-resistant strain was also observed despite only 38% amino acid similarity between these two toxins.

A study was conducted to analyze the influence of midgut-protein-preparative and ligand binding steps on the toxin binding features of a high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins. Total protein was determined using the bicinchoninic acid technique while midguts were homogenized in ice-cold buffer volumes characterized by mannitol and a Tris base. Results suggested that the toxins act through a common receptor and a common mode of action in M. sexta.

Research was conducted to examine the synergism between Bacillus thuringiensis spores and toxins against resistant and susceptible diamondback moths, Plutella xylostella. Results demonstrate that B thuringiensis toxins combined with spores can be toxic despite the fact that the toxins and spores possessed little or no independent toxicity. Findings also indicate that the extent of synergism between toxins and spores of B thuringiensis varies according to insect strain, spore type, the set of toxins, the concentration of toxins and spores and other materials.

The brush border membrane vesicles (BBMVs) from larvae of Manduca sexta, a tobacco hornworm, have two streptavidin-binding proteins that are ca. 120 and 85 kDa. The binding can be prevented by incubating BBMVs with Bacillus thuringiensis, which is biotin-binding. The toxins bind to pyruvate carboxylase, a biotin-containing enzyme, and biotinylated bovine serum albumin and ovalbumin rather than to their nonbiotinylated compounds. The biotin binding site is present in domain III of the toxin and has a sequence similarity to those of streptavidin, avidin, and a biotin specific monoclonal antibody.

The Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis is active against the diamondback moth and is also active against moths that are resistant to the Cry1Ac toxin. Although some insects are becoming resistant to other strains of Bacillus thuringiensis, resistance to israelensis has not been reported.

Researchers have constructed a hybrid of the Bacillus thuringiensis Cry1Ba and Cry1Ia proteins that is toxic to coleoptera such as the Colorado potato beetle. Normally, the Cry1 proteins are only toxic to lepidoptera.

The Cry19A polypeptide from Bacillus thuringiensis subsp. jegathesan may be effective against Culex quinguefasciatus mosquitoes that are resistant to B. thuringiensis subsp. israelensis toxins. There are no field reports of such resistance as of April, 2001, but laboratory studies show that resistance can develop.

Two mannose phosphate isomerase isoenzymes appear to confer resistance to the Bacillus thuringiensis Cry1A toxin in Plutella xylostella. This is the only insect known to be resistant to B. thuringiensis Cry1A toxin in the field, but others have demonstrated potential resistance in the laboratory.

The different methods are evaluated to quantify Cry1Ab toxin obtained from Bacillus thuringiensis (Berliner). The standardization of Cry1Ab production and quantification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry) has improved data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.

The effect of midgut juice isolated from third-instar Manduca sexta on the activity of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Ea, and R233A was studied. Results conclude that it increased the membrane depolarization ability of Cry1Ab with no effect on other mutant.

A study was conducted to examine the role of Trp residues in the structure and function of Cry1Ab toxin, produced by Bacillus thuringiensis. The results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced toxicity against Manduca sexta.

A study was conducted on the rate of pore formation by the trypsin-activated Cry1Aa in the presence of a variety of protease inhibitors with Manduca sexta midgut brush border membrane vesicles, to investigate whether membrane protease are involved in the activity of Bacillus thuringiensis insecticidal toxin. It was observed that, once activated, Cry1Aa is completely functional and does not require further proteolysis.

Thirteen of the most common lepidoteran-specific Cry proteins of Bacillus thuringiensis is tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. It was observed that E. insulana is susceptible to Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry1Ac.

It is shown in the study that cry19Aa in bacillus thuringiensis is enhanced in toxicity for aedes aegypti. This is the first time toxicity in a cry toxin is increased by protein engineering.