Search

In order to understand how lepidopteran insects react physiologically to Bacillus thuringiensis crystal toxin ingestion, transcriptional profiling of Choristoneura fumiferana larvae exposed to sublethal doses of Cry1Ab protoxin were monitored using a C. fumiferana-specific cDNA microarray derived from a protoxin-specific subtractive library. Differential gene expression occurred primarily between 2 and 5 h postingestion. Metabolic enzymes such as lipases and proteases were generally repressed, whereas genes involved in detoxification, immune system regulation or general stress response were upregulated. A similar protoxin-specific transcriptional pattern was also observed with Manduca sexta larvae, using three upregulated genes (serpin, cytochrome P450 and carboxyl/cholinesterase) and one downregulated gene (beta-glucosidase), suggesting that a susceptible larval response to Cry toxin exposure might be universal among lepidopterous insects.

Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation. DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources.

Bacillus thuringiensis is a microbial control agent active against Choristoneura fumiferana, a lepidopteran defoliator of North American forests. Although the B. thuringiensis insecticidal crystal protoxins have a relatively narrow host range, there is concern about their impact on non-target species where intoxication effects may not be overt. Larval toxicity effects can be assessed at the molecular level by determining altered transcriptional profiles in response to sublethal protoxin exposure in sensitive insects. Subtraction hybridization libraries were created using two larval populations, control and protoxin-fed and were characterized by sequencing 1091 clones. Differential mRNA expression of selected clones, as measured by quantitative polymerase chain reaction, identified a number of metabolic and stress-related genes that were either transcriptionally enhanced or repressed after protoxin exposure.

A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.

The role played by alpha-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that alpha-helix 4 plays a crucial role in the mechanism of pore formation.

Bacillus thuringiensis and recombinant Escherichia coli proteinaceous protoxins were subject to proteolysis and analyzed by capillary electrokinetic chromatography. Three resulting toxins (65 kDa) were baseline-resolved within 22 min using a 10 mM borate, pH 11 separation buffer consisting of 25 mM sodium dodecyl sulfate (SDS) and 30 mM phytic acid. The toxins displayed differential interactions with the SDS and phytic acid phases to effect their separation. The ion-pairing interaction between the analyte and phytic acid was also useful in preventing adsorption to the capillary walls and thus enhanced separation resolution and efficiency. The use of electrokinetic chromatography allows achievement of the separation in a significantly shorter time than conventional high-performance liquid chromatography (HPLC) using a diethylaminoethyl (DEAE) weak-anion exchanger., (Copyright 2004 WILEY-VCH Verlag GmbH & Co.)

The binary Bacillus thuringiensis PS149B1 insecticidal crystal (Cry) protein is comprised of two components, Cry34Ab1, a 14-kDa protein, and Cry35Ab1, a 44-kDa protein, the combination of which forms a novel binary toxin active on western corn rootworm larvae. The permeabilizing behavior of the native binary toxin and its two individual components expressed as recombinant proteins was studied using calcein efflux determination in liposomes and by ion channel activity measurements in planar lipid bilayers (PLBs). Data obtained with solubilized native PS149B1 binary protein revealed it to be a pore-forming toxin that can permeabilize liposomes and form ion channels ( approximately 300-900 pS) in PLBs at pH 5.5 but not pH 9.0. The 14-kDa component of the toxin also formed ion channels ( approximately 15-300 pS) at pH 5.5 but did not insert easily in PLBs. While the 44-kDa moiety did seldomly form resolvable ion channels ( approximately 15-750 pS) in PLBs, it did destabilize the membranes. It showed pH-dependent truncation to a stable 40-kDa protein. The purified 40-kDa truncated product formed channels ( approximately 10-450 pS) in PLBs at pH 5.5. At that same pH, while a 3:1 molar mixture (14:44 kDa) of the individual components of the toxin induced channel activity that resembled that of the 14-kDa component alone, the 3:1 molar mixture of the 14-kDa component and 40-kDa truncated product induced channel activity ( approximately 20-800 pS) similar to that of PS149B1 in planar lipid bilayers. We conclude that the overall membrane permeabilization process of Cry34Ab1/Cry35Ab1 is a result of ion channel formation.

The objective of the present work was to create an active Cry1Aa toxin showing enhanced resistance to degradation by spruce budworm (Choristoneura fumiferana) midgut proteases by mutating potential chymotrypsin and trypsin sites. Fourteen Cry1Aa mutants were created in an Escherichia coli-Bacillus shuttle vector and expressed in a crystal minus Bacillus thuringiensis host. Using spruce budworm gut juice, commercial bovine trypsin and chymotrypsin we performed protease resistance assays with Cry1Aa wild type and mutant toxins. Although many mutants showed little or no change, several mutants showed a > 2-fold increase (R543S, R566G, and F570S) up to a > 4-fold increase in toxicity (F576S), in bioassay studies against C. fumiferana. The in vitro protease resistance assay results indicated a possible involvement of other gut juice components in toxin overdigestion.

Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.

Dynamic light scattering and surface plasmon resonance techniques were used to investigate the influence of ionic strength, buffer composition and pH on the multimerization of trypsin-activated Cry1Ac and Cry1C toxins over time and the subsequent effects of the different multimers on receptor binding models. In carbonate buffer at pH 10.5, Cry1Ac and Cry1C assumed a monomeric state. After 24 h, a complete conversion of monomeric toxin to a dimeric or trimeric form was observed only for Cry1Ac under low ionic strength condition. Cry1C and Cry1Ac in high ionic strength buffer remained monomeric. Substitution of CAPS pH 11 for carbonate buffer suppressed this Cry1Ac oligomerization effect. Once Cry1Ac toxin was in an aggregated form, increases in ionic strength failed to revert the aggregated toxin back to a monomeric form. Monomeric Cry1Ac bound to a purified 115 kDa aminopeptidase N receptor from Manduca sexta in a 2:1 molar ratio thus confirming the existence of two binding sites on this receptor. Binding rates of dimeric or higher aggregated Cry1Ac toxin forms were different from those generated using the monomeric form and could not be fitted to existing binding models. In summary, our results confirm that the M. sexta 115 kDa aminopeptidase N receptor possesses two Cry1Ac binding sites. They further suggest that although high pH and low salt conditions promote Cry1Ac aggregation, this observation cannot be applied universally to other members of the Cry family.

We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

Influence of domain I exchange on the stability and production of Bacillus thuringiensis Cry1 protoxins as well as on the shape of inclusion and toxicity to Spodoptera exigua and Plutella xylostella larvae was investigated. Chimeric genes were prepared by exchanging the regions coding for domain I between Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The AcCC chimera accumulated into bipyramidal inclusion bodies, whereas CEE produced round-shaped inclusion bodies, and ECC and AaEE protoxins produced small granules. AbEE and EAaAa did not produce any inclusion body and were visualized by immunodetection only. AcCC, CEE, ECC, and AaEE were stable to trypsin, whereas AbEE and EAaAa were not. Bioassays showed that the chimeras were not toxic in vivo. However, S. exigua larvae fed with the activated AcCC toxin displayed a lower growth rate.

The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.

Whole-crystal preparations from strains HD-1 and HD-133, activated Cry1Ab and Cry1C toxins as well as Cry1Aa, Cry1Ac, Cry1D, and Cry2Aa protoxins were tested for toxicity to 2nd-instar larvae of the diamondback moth, Plutella xylostella. Mortality data recorded after 2 and 5 days provided different results that were related to differential rates of solubilization, activation, and degradation of insecticidal crystal proteins. The two most active proteins are Cry1Ab and Cry1C, which are both present in HD-133. The Cry1Ab protoxin is activated within 2 days, whereas activation of the Cry1C protoxin occurs between 2 and 5 days. HD-133 is more active than HD-1 immediately after infection and remains toxic over 5 days owing to the sequential activation of its crystal components. Solubility properties of crystals and rates of activation of protoxins influence the overall toxicity of HD-1 and HD-133 to the diamondback moth.

The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought.

The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique. Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner. In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect. Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose. On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0-1.2 nm. In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.

The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with KD ranging from 41 to 95 nM and a lower affinity site with KD in the 325 to 623 nM range. N-acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86Rb+ release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86Rb+ release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86Rb+ release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.

A purified, GPI-linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33-1.7 nM) than in receptor-free membranes. The non-toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CrylAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current-voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.

Disulfide bridges were introduced into CrylAa, a Bacillus thuringiensis lepidopteran toxin, to stabilize different protein domains including domain I alpha-helical regions thought to be involved in membrane integration and permeation. Bridged mutants could not form functional ion channels in lipid bilayers in the oxidized state, but upon reduction with beta-mercaptoethanol, regained parental toxin channel activity. Our results show that unfolding of the protein around a hinge region linking domain I and II is a necessary step for pore formation. They also suggest that membrane insertion of the hydrophobic hairpin made of alpha-helices 4 and 5 in domain I plays a critical role in the formation of a functional pore.

The activated 65 kDa lepidopteran-specific CryIA(a) toxin from the commercially most important strain Bacillus thuringiensis var. kurstaki HD-1 has been investigated by X-ray diffraction and for its ability to form channels in planar lipid bilayers. Its three-dimensional structure has been determined by a multiple isomorphous replacement method and refined at 2.25 A resolution to an R-factor of 0.168 for data with I > 2 delta (I). The toxin is made of three distinct domains. The N-terminal domain is a bundle of eight alpha-helices with the central, relatively hydrophobic helix surrounded by amphipathic helices. The middle and C-terminal domains contain mostly beta-sheets. Comparison with the structure of CryIIIA, a coleopteran-specific toxin, shows that although the fold of these two proteins is similar, there are significant structural differences within domain II. This finding supports the conclusions from genetic studies that domain II is involved in recognition and binding to cell surface receptors. The distribution of electrostatic potential on the surface of the molecule is non-uniform and identifies one side of the alpha-helical domain as negatively charged. The predominance of arginine residues as basic residues ensures that the observed positive charge distribution is also maintained in the highly alkaline environment found in the lepidopteran midgut. Structurally important salt bridges that are conserved across Cry sequences were identified and their possible role in toxin action was postulated. In planar lipid bilayers, CryIA(a) forms cation-selective channels, whose conductance is significantly smaller than that reported for CryIIIA but similar to those of other Cry toxins.

The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.

An optical biosensor technology based on surface plasmon resonance was used to determine the kinetic rate constants for interactions between the CryIA(c) toxin from Bacillus thuringiensis and brush border membrane vesicles purified from susceptible and resistant larvae of diamondback moth (Plutella xylostella). CryIA(c) association and dissociation rate constants for vesicles from susceptible larvae were determined to be 4.5 x 10(3) M-1 s-1 and 3.2 x 10(-5) s-1, respectively, resulting in a calculated affinity constant of 7 nM. CryIE toxin did not kill susceptible or resistant larvae and did not bind to brush border vesicles. Contrary to expectations based on previous studies of binding in resistant P. xylostella, the binding kinetics for CryIA(c) did not differ significantly between susceptible larvae and those that were resistant to CryIA(c). Determination of the number of CryIA(c) receptors revealed an approximately 3-fold decrease in total CryIA(c) receptor numbers for resistant vesicles. These results suggest that factors other than binding may be altered in our resistant diamondback moth strain. They also support the view that binding is not sufficient for toxicity.

A 3 kb DNA region upstream of the toluene degradation (tod) genes, todFC1C2BADEGIH, in Pseudomonas putida F1 (PpF1) was sequenced. Two divergently arranged open reading frames, todR and todX, were identified. A toluene-inducible promoter was localized in front of todX, and the transcription start point was mapped. This promoter is probably responsible for the expression of all tod structural genes. TodX was found to be a membrane protein. Its predicted amino acid sequence (453 residues; M(r) 48,265) exhibits considerable similarity with the FadL protein of Escherichia coli, an outer membrane protein required for binding and transport of long-chain fatty acids. An apparent function of TodX is likely to be involved in facilitating the delivery of exogenous toluene inside the PpF1 cells. The sequence of TodR (100 residues) exhibits extensive homology with the DNA-binding domain of transcriptional activators of the LysR family, but todR was found to have a negligible role in tod gene regulation.

The Bacillus thuringiensis crylA(a) and crylA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CrylA(c) is highly active against cultured Choristoneura fumiferana cells (CF-1) whereas CrylA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis revealed that the CrylA(c) toxicity-determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258-358 and 450-510) were able to confer toxicity, although at lower levels, and one region (358-450) was present where progressive substitutions of crylA(a) with crylA(c) sequences had no effect. Exchanging the non-homologous N-terminal regions of CrylA(c) with CrylE suggested that the N-terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CrylA(b) but shows an 800-fold increase in toxicity to CF-1 cells relative to that shown by CrylA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity-determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450-620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.

In order to study the kinetics of binding between membrane vesicle surface receptors to the lepidopteran insecticidal toxins from Bacillus thuringiensis using surface plasmon resonance, we have developed a technique to immobilize membrane vesicles purified from the brush border of dissected guts from the lepidopteran insect pest Choristoneura fumiferana. Two methods using immobilized immunoglobulins against either avidin or biotin were successful in achieving stable immobilization of the vesicles (> 1.5 h). Specificity of the immobilized receptors exposed on the vesicle surface was demonstrated, in part, by the inability of bovine serum albumin to bind to the immobilized brush border membrane vesicles. Homologous and heterologous competition experiments further demonstrated specific binding of trypsin-activated CryIA(c) toxin to the cell-surface receptors on the vesicles. Kinetic rate constants for activated cryIA(b) toxin binding to brush border vesicles were determined, revealing the presence of a high-affinity receptor on the surface of the immobilized brush border membrane vesicles.

Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25-35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.

The lipoprotein I gene (oprI) of Pseudomonas aeruginosa PAO1 was cloned and sequenced. A high degree of homology was found between our cloned PAO1 gene sequence and two published oprI sequences. Specific oligonucleotides were designed to amplify the oprI gene by the polymerase chain reaction (PCR). The potential of either the complete gene sequence or the specific oligonucleotide primers as a tool for rapid strain identification was directly assessed against bacterial colonies by PCR or against purified genomic DNA by Southern blot analysis, using a number of representative strains within the Pseudomonadaceae. The oprI gene was found to be well conserved within RNA group I.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.