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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.

[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 novel cry gene cry32Aa of Bacillus thuringiensis subsp. yunnanensis is described. The gene was introduced into E. coli and the resulting protein was weakly toxic against the diamondback moth Plutella xylostella but was not toxic to other moths or mosquitoes.

All strains of Bacillus thuringiensis appear to have genes for the Cyt toxins, which are active against dipteran, lepidopteran, and coleopteran larvae. DNA and protein sequencing reveals a high degree of conservation in this toxin.

The gene cyt1Aa which is one of the genes in the complex that determines the mosquito larvicidity of Bacillus thuringiensis subsp. israelensis is reported. The gene cyt1Aa is recloned without the artifact to retest its toxicity against Aedes aegypti larvae and host of E. coli cells.

Cry11Aa helix [alpha]-3 mutants impaired in oligomerization and toxicity against Aedes aegypti is characterized, showing that oligomerization of Cry11Aa is important for toxin action. CytAa from Bacillus thuringiensis has not recovered the insecticidal activity of Cry11Aa mutants.

The ability of the cadherin fragments obtained from Anopheles gambiae to synergize the toxicity of Bacillus thuringiensis subsp. israelensis Cry4Ba against Aedes aegypti larvae is discussed. The fragments are demonstrated to be highly syngergizing and hence are widely used for the purpose.

The differences in activation between spores from strains of Bacillus thuringiensis subsp. israelensis with and without the toxin-encoding plasmid pBtoxis are demonstrated. The strain bearing pBtoxis showed a significantly better germination rate following alkaline activation.

The resistance of greenhouse-selected strains of the cabbage looper, Trichoplusia ni, to Bacillus thuringiensis subsp. kurstaki is countered by a hybrid strain of Bacillus thuringiensis and genetically modified toxins Cry1AMod and Cry1AcMod, which has lacked helix [alpha]-1. The resistance to Cry1AbMod and Cry1AcMod is lesser than resistance to native toxins Cry1Ab and Cry1Ac.

A Bacillus thuringiensis enhancin-like gene (bel) encoding a protein (Bel) that has 20 to 30% identity to the viral enhancin protein and 95% identity to bacterial enhancin-like proteins is described. The results have implied that Bel toxicity enhancement has depended on the destruction of midgut peritrophic matrix (PM) and intestinal mucin (IIM), similar to the case with viral enhancin.

Four Bacillus thuringiensis [delta]-endotoxins, Cry3A, Cry4Aa, Cry11Aa, have displayed low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab is essentially nontoxic except at high rates and these effects are shown by using exhaustive buffer-based controls.

Parasporal bodies of arsenal toxin Cry10Aa are formed by cloning the whole Cry10Aa operon under the control of the cyt1A promoter and the STAB-SD stabilizer sequence. The studies have shown that Cry10Aa is as toxic as most of the other Bacillus thuringiensis subsp. israelensis toxins acting separately and in synergism with the Cyt1A toxin.

The fragment containing the three most C-terminal cadherin repeats (CR) from the cadherin of the western corn rootworm has bound toxins and has enhanced Cry3 toxicity to larvae of naturally susceptible species. The region spanning CR8 to CR10 of the WCRW cadherin is cloned and expressed in Escherichia coli and this cadherin fragment has enhanced the toxicities of Bacillus Thuringiensis Cry3Aa and Cry3Bb toxins to Colorado potato beetle (CPB) and rootworms.

The design, construction, and analysis of a set of crybodies were carried out to understand the effect of loop substitution on toxin stability and specificity of Bacillus thuringiensis. The results showed that the domain II apical loops play important role as determinants of binding specificity to cadherins, a major class of toxin receptors.

A modified coleopteran-active Bacillus thuringiensis toxin (Cry3A) toxin (mCry3A) is designed with a chrymotrypsin/cathepsin G site in domain I which displays greater biological activity than Cry3A. The studies have shown that the mCry3A toxicity for corn rootworm larvae is used to the introduction of a chymotrypsin/cathepsin G site, which increased cleavage and binding of the activated toxin to midgut cells.

The physiology of binding of the cry proteins to the receptors in the insect cells and the insecticidal properties of these toxins from Bacillus thuringiensis are investigated.