Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ϳ1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ϳ30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.Xenorhabdus and Photorhabdus spp. are two bacterial genera belonging to the family Enterobacteriaceae, known to be associated with entomopathogenic nematodes (1-4) These bacteria represent potential sources for new genes encoding potent insecticidal toxins that could be put into plants as alternatives to Bacillus thuringiensis genes (5). Gene sequence analysis of Xenorhabdus and Photorhabdus bacteria show that these organisms contain a family of related toxin complex (tc) 2 genes located at different loci (6 -9). The toxin complexes are composed of three different classes of protein components, which, according to ffrench-Constant et al. (10,11), can be categorized as class A, B, and C proteins based upon sequence similarity and size. Class A proteins are very large, having a molecular mass of ϳ280 kDa. Class B proteins are ϳ170 kDa, and class C proteins are ϳ110 kDa. There are many different varieties of class A, B, and C proteins in both Gram-negative and Gram-positive bacteria (12-15).From earlier studies, it has been suggested that class A proteins harbor the cytotoxic effects of the Tc toxins, whereas class B and C proteins modulate and enhance the toxicity of class A proteins (16). However, recently, we elucidated the molecular mechanism of the Photorhabdus luminescens Tc complex, which consists of the class A protein TcdA1, the class B protein TcdB2, and the class C protein TccC3 or TccC5 (17). These studies revealed that the class C proteins harbor the biological activity. It was shown that TccC3 and TccC5 are ADP-ribosyltransferases, which target the actin cytoskeleton by modification of actin and Rho GTPases, respectively (17). Moreover, these studies su...

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Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.

Cotton plants were genetically modified through the introduction of a synthetic gene that encodes a Bacillus thuringiensis insecticidal protoxin referred to as Cry1F(synpro). This protoxin is a chimeric, full-length delta-endotoxin of 130 kDa, comprised of the core toxin of Cry1Fa2 protein and parts of the nontoxic portions of Cry1Ca3 and Cry1Ab1 proteins, all of which originated from Bacillus thuringiensis. The Cry1F(synpro) expressed in cotton plants confers resistance to lepidopteran pests. The current study was conducted to characterize the Cry1F(synpro) protein expressed in the transgenic cotton event 281-24-236. Results showed that the full-length Cry1F(synpro) produced in the transgenic cotton plants was sensitive to the host cell protease cleavage, resulting in a truncated, biologically active form (core toxin) with an apparent molecular mass of 65 kDa. This truncated toxin was purified by immunoaffinity chromatography from the cotton leaf extract. N-terminal sequencing, peptide mass fingerprinting by MALDI-TOF MS, and internal peptide sequencing by MS/MS confirmed the identity of the truncated core toxin of Cry1F. The mechanism of truncation was explored with Cry1F(synpro) derived from a recombinant Pseudomonas fluorescens. The transgenic cotton-produced Cry1F showed equivalent insecticidal activity to that of Pseudomonas fluorescens-derived Cry1F.