Characterization of ISBth4, a functional new IS231 variant from Bacillus thuringiensis MEX312.

A new insertion variant of IS231 family, designated ISBth4, was identified from Bacillus thuringiensis MEX312. ISBth4 is 2046bp in length and is delimited by two 17bp inverted repeats (IR) with one mismatch, flanked by two perfect 11bp direct repeats (DR). ISBth4 contains two open reading frames (ORFs), ORF1 and ORF2, which encode 72 and 473 amino acids, respectively. Multiple sequence alignments revealed that the potential transposase of ISBth4 contained five conserved domains N1, N2, N3, C1 and C2 that are similar to other IS231 elements; and the typical catalytic triad D(N2)-70-D(N3)-150-E(C1) and Y(2)R(3)E(6)K motifs as hallmarks of IS4 elements. Comparison of the amino acids of the potential ISBth4 transposase with those from other publicly available B. cereus group IS231 elements revealed a close similarity with ISBce7 (94% identity), ISBce5 (90%), IS231Y (89%) and ISBce8 (86%), and lower similarity to IS231N (49%), IS231M (48%) and ISBce12 (40%). Phylogenetic analysis of the evolutionary relationships between ISBth4 and the other IS231 elements showed that ISBth4 is more closely related to the IS231 sequences isolated from B. cereus strains than those from B. thuringiensis strains. In vivo transposition activity of ISBth4 was discovered in a mutant B18 from a MEX312 background, indicating that it is a functional insertion sequence in its B. thuringiensis natural host.

Peritrophic membrane contribution to Bt Cry delta-endotoxin susceptibility in Lepidoptera and the effect of Calcofluor.

In an effort to determine a reason for differing susceptibilities to Bacillus thuringiensis (Bt) Cry delta-endotoxins amongst lepidopteran species, the peritrophic membrane (PM), and a number of agents that target the PM, were investigated to determine their effect on the efficacy of Bt toxins. In particular Calcofluor is able to disrupt the PM that acts as a barrier to microorganisms. Although Bt toxins have been shown to traverse the PM in some lepidopteran species, new data shows that toxins can bind the PM. Lepidopteran larval PMs also vary in thickness and composition that may determine the passage of Bt toxins. In non-susceptible insects the toxin associates with PM proteins and frass and is thought to be retained by the PM and then excreted before binding to the exposed target midgut membrane. However, the addition of Calcofluor to Bt toxins at an LC50 for the recipient species did not result in an increase in the efficacy of the toxin. It is evident that Calcofluor does disrupt the PM but the toxin preferentially binds PM fragments and is excreted instead of binding the exposed target midgut membrane, therefore having little toxic effect. This study therefore concludes that Calcofluor is not as suitable as other toxin enhancing agents such as chitinase.

Investigating the properties of Bacillus thuringiensis Cry proteins with novel loop replacements created using combinatorial molecular biology.

Cry proteins are a large family of crystalline toxins produced by Bacillus thuringiensis. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create "Crybodies." Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins--a major class of toxin receptors--since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.

Molecular characterization of virulence defects in Bacillus thuringiensis mutants.

Sequence analysis of a virulence-attenuated Bacillus thuringiensis signature tagged mutant 6F8 led to the identification of an 18 182 bp locus encoding 29 potential protein-coding ORFs. Thirteen of the 29 putative ORFs were found to share extensive homology with genes on plasmid pE33L466 of the pathogenic Bacillus cereus E33L strain. Nine ORFs were not only found in a cluster, but also in the same gene order, in both organisms. A number of mobile elements, including a transposon Tn4430, a novel IS231 family insertion sequence ISBth4 and various phage-related proteins, were found flanking this conserved gene cluster. These features of the 6F8 locus suggested that it might have undergone several DNA insertions from different sources by horizontal gene transfer. Transcriptional analyses of the 6F8 locus revealed that ORFs 1-23 were cotranscribed as a single transcript. Null mutants were constructed to investigate the function of the sequences flanking the signature-tagged mutagenesis insertion sites. Competition assays performed with the wild-type and null mutants demonstrated that the Tn4430 transposon element plays an important role in the full virulence of B. thuringiensis during Manduca sexta infection. This study provides the first experimental evidence that a Tn4430 family transposon is directly associated with B. thuringiensis virulence.

Expression and insecticidal activity of Yersinia pseudotuberculosis and Photorhabdus luminescens toxin complex proteins.

Photorhabdus luminescens toxin complex (Tc) has been characterized as a potent three-component insecticidal protein complex. Homologues of genes encoding P. luminescens Tc components have been identified in several other enterobacteria and in Gram-positive bacteria, showing these genes are widespread in bacteria. In particular, tc gene homologues have been identified in Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis and may have a role in Y. pestis evolution. Y. enterocolitica tc genes have been shown to be active against Manduca sexta larvae. Here, we demonstrate that expression optimization is essential to obtain bioactive P. luminescens Tc proteins and demonstrate that TcaAB and TcdB + TccC are stand-alone toxins against a M. sexta insect model. Moreover, we report that Y. pseudotuberculosis IP32953 Tc proteins are also toxic to M. sexta larvae but do not cross-potentiate as P. luminescens Tc components.

Role of receptors in Bacillus thuringiensis crystal toxin activity.

Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death.

The mutation R(423)S in the Bacillus thuringiensis hybrid toxin CryAAC slightly increases toxicity for Mamestra brassicae L.

Bacillus thuringiensis Cry1Ac toxin is 100 times less toxic than Cry1C to Mamestra brassicae. An R(423)S mutation abolishes Cry1Ac toxin proteolysis in M. brassicae gut juice but does not increase its toxicity to this insect. The CryAAC hybrid toxin (1Ac/1Ac/1Ca) is toxic to M. brassicae but is susceptible to gut protease digestion at the R(423) residue. Accordingly we have investigated the effect of the R(423)S mutation in CryAAC on its toxicity for M. brassicae and Pieris brassicae. Bioassays demonstrated that the R(423)S mutation slightly increased the toxicity of CryAAC for M. brassicae by having a significantly inhibitory effect on the growth of surviving larvae. The mutant hybrid was still highly toxic to P. brassicae. Features of CryAACR(423)S such as, (1) stability in M. brassicae gut juice and (2) crystal solubility were investigated. Computer simulations suggest that a possible major increase in flexibility in the CryAAC loop beta7/beta8 (G(391)-P(397)) caused by the R(423)S substitution could be a reason for the increase in M. brassicae toxicity.

Discovery of Bacillus thuringiensis virulence genes using signature-tagged mutagenesis in an insect model of septicaemia.

Transposon Tn917 was used to identify Bacillus thuringiensis genes required for virulence and survival in a Manduca sexta (tobacco hornworm) septicaemia model. Uniquely tagged transposons, n = 72, were constructed and used to generate 1152 insertion mutants. Sixteen pools of 72 mutants were screened in the infection model, and 12 virulence-attenuated mutants were unable to survive the infection. Analysis of the mutated DNA sequences implicated an arsR family transcriptional regulator, a histone-like DNA-binding protein, a transposon, and several sequences of unknown function in B. thuringiensis pathogenesis.

How to kill a mocking bug?

All metazoans have evolved means to protect themselves from threats present in the environment: injuries, viruses, fungi, bacteria and other parasites. Insect protection includes innate physical barriers and both cellular and humoral responses. The insect innate immune response, best characterized in Drosophila melanogaster, is a rapid broad response, triggered by pathogen-associated molecular patterns (PAMPs) recognition, which produces a limited range of effectors that does not alter upon continued pathogen exposure and lacks immunological memory. The Drosophila response, particularly its humoral response, has been investigated by both low and high-throughput methods. Three signalling pathways conserved between insects and mammals have been implicated in this response: Toll (equivalent to mammalian TLR), Imd (equivalent to TNFalpha) and Hop (equivalent to JAK/STAT). This review provides an entry point to the insect immune system literature outlining the main themes in D. melanogaster bacterial pathogen detection and humoral and cellular immune responses. The Drosophila immune response is compared with other insects and the mammalian immune system.

Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin.

Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis.

Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the alphaD-beta4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on alphaE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on alphaA, alphaB, alphaC, alphaD and the loops between alphaA and alphaB, alphaD and beta5, beta6 and beta7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.

A ferric dicitrate uptake system is required for the full virulence of Bacillus cereus.

Bacillus cereus is an opportunistic human pathogen of increasing prevalence. Analysis of the Bacillus cereus genome sequence identified a potential ferric dicitrate uptake system. The three-gene operon was confirmed to be negatively regulated by the ferric uptake repressor (Fur). The Fec operon was genetically silenced using the integration suicide vector pMUTIN4. The mutant strain displayed no growth defect under iron-limited conditions but was unable to grow on ferric citrate as a sole iron source. The virulence of the mutant strain was attenuated in a lepidopteran infection model, highlighting the importance of iron uptake systems to the virulence of B. cereus and the potential of these systems to act as targets for novel antimicrobial agents.

Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications.

Cry4Ba, isolated from Bacillus thuringiensis subsp. israelensis, is specifically toxic to the larvae of Aedes and Anopheles mosquitoes. The structure of activated Cry4Ba toxin has been determined by multiple isomorphous replacement with anomalous scattering and refined to R(cryst) = 20.5% and R(free)= 21.8% at 1.75 Angstroms resolution. It resembles previously reported Cry toxin structures but shows the following distinctions. In domain I the helix bundle contains only the long and amphipathic helices alpha3-alpha7. The N-terminal helices alpha1-alpha2b, absent due to proteolysis during crystallisation, appear inessential to toxicity. In domain II the beta-sheet prism presents short apical loops without the beta-ribbon extension of inner strands, thus placing the receptor combining sites close to the sheets. In domain III the beta-sandwich contains a helical extension from the C-terminal strand beta23, which interacts with a beta-hairpin excursion from the edge of the outer sheet. The structure provides a rational explanation of recent mutagenesis and biophysical data on this toxin. Furthermore, added to earlier structures from the Cry toxin family, Cry4Ba completes a minimal structural database covering the Coleoptera, Lepidoptera, Diptera and Lepidoptera/Diptera specificity classes. A multiple structure alignment found that the Diptera-specific Cry4Ba is structurally more closely similar to the Lepidoptera-specific Cry1Aa than the Coleoptera-specific Cry3Aa, but most distantly related to Lepidoptera/Diptera-specific Cry2Aa. The structures are most divergent in domain II, supporting the suggestion that this domain has a major role in specificity determination. They are most similar in the alpha3-alpha7 major fragment of domain I, which contains the alpha4-alpha5 hairpin crucial to pore formation. The collective knowledge of Cry toxin structure and mutagenesis data will lead to a more critical understanding of the structural basis for receptor binding and pore formation, as well as allowing the scope of diversity to be better appreciated.

Bacillus cereus Fur regulates iron metabolism and is required for full virulence.

A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a beta-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The Deltafur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.

Discrimination of pathogenic clinical isolates and laboratory strains of Bacillus cereus by NMR-based metabolomic profiling.

Six different Bacillus cereus strains were selected from two different ecotypes: (1) three commonly used laboratory strains that are considered avirulent, and (2) three clinical isolates from meningitis patients. Screening of genomic DNA for the presence of genes encoding known toxins gave no candidate genes that were unambiguously able to distinguish between the two groups. However, the application of multivariate pattern-recognition methods to metabolite profiles derived from the different strains using 1H nuclear magnetic resonance spectroscopy (metabolomics) was able to classify the different profiles. The two different ecotypes were clearly separated on the basis of their metabolite profiles, showing that it is possible to use metabolomic methods to classify pathogens on the basis of their expressed physiology, even when it is not possible to infer a direct mechanistic link to specific virulence factors. This metabolomic approach could also have a wide range of possible applications in both general microbiology and microbial ecology for distinguishing and identifying different functional/physiological ecotypes of bacterial strains or species.

Display of biologically functional insecticidal toxin on the surface of lambda phage.

The successful use of Bacillus thuringiensis insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.

The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures.

Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual structures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccharides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual difucosylated cores. Cross-ring fragment ions and 'internal' fragment ions observed in the CID spectra, showed that these fucoses are found at the 3-position of proximal GlcNAc and at the 3-position of distal GlcNAc in the chitobiose unit. The latter substitution has only been previously observed in nematodes. In addition, these core structures can be decorated with novel fucosylated antennae composed of Fucalpha(1-3)GlcNAc. Key fragment ions revealed that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N-glycan (Man(3)GlcNAc(2)), commonly found on other insect glycoproteins, is the predominant oligosaccharide found at the remaining N-glycosylation site (Asn609).

Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin.

The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta. The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to 120 kDA APN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor combinations. We analyzed the various glycans on the 120 kDA APN using carbohydrate compositional analysis and lectin binding. The data indicate that in the average APN molecule, 2 of 4 possible N-glycosylation sites are occupied with fucosylated paucimannose [Man(2-3)(Fuc(1-2)GlcNAc(2)-peptide] type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal "stalk" region of the protein. It is likely that 5-6 of the 13 sites are occupied, probably with simple [GalNAc-peptide] type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc-rich, is the most likely binding site for Cry1Ac.

Interaction of cytolytic toxin CytB with a supported lipid bilayer: study using an acoustic wave device.

An acoustic technique was used to monitor the interaction of the pore-forming cytolytic toxin CytB with a positively charged supported lipid bilayer. The acoustic device, which is based on a waveguide geometry, is sensitive to changes in the mass of the supported bilayer. The specificity of the interaction, rate and extent of the association, reversibility and effect of previous depositions of toxin were investigated. The CytB was found to bind irreversibly to the lipids at all fractional coverages even when the protein-to-lipid ratio was high enough to imply that the protein was associating with the external surface of the bilayer. The CytB formed stable structures with the bilayer at high protein surface concentrations and did not appear to disrupt the bilayer in the manner of a detergent. The rate of association with the bilayer was found to be directly proportional to the solution concentration of CytB at higher concentrations but appeared to be low at a CytB solution concentration of 5 microg mL(-1), leading to relatively low amounts of CytB being associated with the bilayer.

High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector.

The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the recombinant production of a wide variety of proteins. Initial success with this system was greatly facilitated by the development of versatile expression vectors that were almost exclusively based on the strong, tightly regulated promoter of the P. pastoris major alcohol oxidase gene ( AOX1 ). For example, pIB4 is an Escherichia coli - P. pastoris shuttle vector that also uses the AOX1 promoter to allow intracellular expression of endogenous and foreign genes in the latter organism. Since the eukaryotic advantages of P. pastoris would be best harnessed through the secretory targeting of the recombinant proteins, we modified the pIB4 vector by adding the Saccharomyces cerevisiae alpha-factor secretion signal immediately upstream of its multiple cloning site. Here we describe the construction of this modified vector, pIB4alpha, and its successful use for the high-level expression and secretion of a functional single-chain antibody fragment (scFv), C6.5, which targets p185(HER-2), a cell-surface glycoprotein overexpressed in about 30% of human breast and ovarian cancers. The PCR strategy used for the subcloning of the C6.5 construct into pIB4alpha also introduced a short DNA sequence coding for a C-terminal hexahistidine tag, which allowed subsequent purification of the secreted scFv, by immobilized-metal-affinity chromatography, to a yield of 70 mg x l(-1) of shake-flask culture. In conclusion, our results suggest that the secretion expression vector pIB4alpha not only complements the original pIB4 vector for intracellular expression in P. pastoris, but might also constitute an attractive alternative to the commercially available secretion expression vectors.