Resistance to root-knot nematode in tomato roots expressing a nematicidal Bacillus thuringiensis crystal protein

Our laboratory has demonstrated previously that Bacillus thuringiensis (Bt) crystal (Cry) proteins present in the Cry5 and Cry6 subclades intoxicate free-living nematodes. In this study, we tested whether the expression of nematicidal Cry6A in transgenic plants provided protection against plant-parasitic nematodes. As bacterial codon usage is incompatible with expression in plants, two different codon-modified cry6A genes were synthesized for expression in plants. One was designed by maintaining codon diversity whilst removing codons not common in plants, and the other was designed by selecting the optimal codon for each amino acid based on the Arabidopsis genome. Both versions of the cry6A gene, driven by the constitutive cauliflower mosaic virus 35S promoter, were introduced into tomato roots via Agrobacterium rhizogenes . Although both were found to express Cry6A protein, the codon diversity gene generated superior expression. These Cry6A-expressing roots were then challenged with root-knot nematode, Meloidogyne incognita . Three different infection parameters were compared between Cry6A-expressing roots and control roots transformed with empty vector or green fluorescent protein (GFP). These data demonstrated that M. incognita was able to ingest the 54-kDa Cry6A, and that Cry6A intoxicated the parasitic nematode, as indicated by a decrease in progeny production of up to fourfold. These results indicate, for the first time, that a Bt Cry protein can confer plant resistance to an endoparasitic nematode, and that Cry proteins have the potential to control plant-parasitic nematodes in transgenic plants.     

苏云金芽孢杆菌及其杀虫晶体蛋白 作用机制的研究进展

综合叙述了苏云金芽胞杆菌Bacillus thuringiensis和杀虫晶体蛋白的作用机制及在不同水平上解释这些机制的一些流行模型和有关亚分子结构的作用。     

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism

Summary The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 513 and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.     

Heterologous expression of a mutated toxin gene from Bacillus thuringiensis subsp. tenebrionis

Using oligonucleotide probes we have isolated a DNA fragment encoding an insecticidal toxin of the coleopteran specific Bacillus thuringiensis subsp. tenebrionis. The gene was altered by site directed mutagenesis at its 5'-end and adapted for general cloning and expression purposes with a linker including a start codon and new restriction sites. The constructs were inserted into several vector plasmids and expressed in Escherichia coli. Expression E. coli was strongly enhanced by the lac-promoter. A fusion protein with phage MS2-polymerase was produced together with a 67 kDa protein also found for normal expression of the toxin gene. Synthesis of the latter protein indicated a second ribosome binding site at the 5'-terminus of the toxin encoding sequence. Toxin-containing proteins were identified by Western blot analysis. The positive cell extracts from E. coli had insecticidal activity on larvae of the Colorado potato beetle. The cloned gene is not homologous to a gene previously cloned by us whose gene products were also toxic to coleopteran larvae.     

Transference of a crystal protein gene from Bacillus thuringiensis and its expression in Bradyrhizobium sp. cells

The plasmid pHT409 that harbours the cryIA(a) gene for the production of a -endotoxin (crystal protein) from Bacillus thuringiensis was transferred into Bradyrhizobium sp. A conjugal transfer system aiming to introduce the plasmid into the Bradyrhizobium sp. host from colonies of an Escherichia coli donor strain (DH5::pHT409) has been developed. As a result exconjugants were obtained in which the transferred plasmid has been detected by both microbiological and electrophoresis techniques. The cryIA(a) gene when inside the new host had a low expression level which was detected by immunoblotting.     

Bacillus thuringiensis crystal protein (δ-endotoxin) gene expression is independent of early sporulation specific functions

Bacillus thuringiensis produces a parasporal insecticidal crystal protein. The correlation between sporulation and crystal protein production inBacillus thuringiensis var.israelensis was studied. The strain was made resistant’against streptomycin (St^R)-Acrystalliferous (Cry^-) cured derivatives and asporogenous acrystalliferous (Spo⁻ Cry⁻) mutants blocked at an early stage of sporulation were isolated. Plasmid transfer experiments were performed between St^R Spo⁺ Cry⁺ (streptomycin sensitive sporogeneous crystalliferous) and St^RR Spo⁺ Cry⁻ and also between St^s Spo⁺ Cry⁺ and St^R Spo⁻ Cry⁻ strains. StR colonies were selected. Insect toxicity was exhibited by the St^R isolates in both the cases. The process of crystal formation is, therefore, independent of early sporulative events.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

The absence of tertiary interactions in a self-assembled DNA crystal structure

DNA is a highly effective molecule for controlling nanometer-scale structure. The convenience of using DNA lies in the programmability of Watson-Crick base-paired secondary interactions, useful both to design branched molecular motifs and to connect them through sticky-ended cohesion. Recently, the tensegrity triangle motif has been used to self-assemble three-dimensional crystals whose structures have been determined; sticky ends were reported to be the only intermolecular cohesive elements in those crystals. A recent communication in this journal suggested that tertiary interactions between phosphates and cytosine N(4) groups are responsible for intermolecular cohesion in these crystals, in addition to the secondary and covalent interactions programmed into the motif. To resolve this issue, we report experiments challenging this contention. Gel electrophoresis demonstrates that the tensegrity triangle exists in conditions where cytosine-PO(4) tertiary interactions seem ineffective. Furthermore, we have crystallized a tensegrity triangle using a junction lacking the cytosine suggested for involvement in tertiary interactions. The unit cell is isomorphous with that of a tensegrity triangle crystal reported earlier. This structure has been solved by molecular replacement and refined. The data presented here leave no doubt that the tensegrity triangle crystal structures reported earlier depend only on base pairing and covalent interactions for their formation.     

DNA fingerprinting reveals significant amounts of genetic variation in a wild raspberry Rubus idaeus population

Establishing the genotypic distribution in natural plant populations is an important part of ecological studies concerning plant growth, reproduction and turn-over. Restriction enzyme-digested DNA samples, isolated from 24 plants of a natural Rubus idaeus population, were analysed with DNA fingerprinting using the M13 repeat sequence as well as a synthetic (AC)/(TG) polydinucleotide as hybridization probes. All the examined samples exhibit unique DNA fingerprint patterns, suggesting that vegetative reproduction may be considerably more restricted in wild R. idaeus populations than previously assumed. By comparison, all samples of the apomictic blackberry species Rubus nessensis, collected on the same location, were completely identical.     

The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins

YhaK is a protein of unknown function found in low abundance in the cytosol of Escherichia coli. DNA array studies have revealed that YhaK is strongly up-regulated by nitroso-glutathione (GSNO) and also displays a 12-fold increase in expression during biofilm growth of E. coli 83972 and VR50 in human urine. We have determined the YhaK crystal structure and demonstrated that in vitro YhaK is a good marker for monitoring oxidative stresses in E. coli. The YhaK protein structure shows a bicupin fold where the two cupin domains are crosslinked with one intramolecular disulfide bond (Cys10 to Cys204). We found that the third cysteine in YhaK, Cys122, is oxidized to a sulfenic acid. Two chloride ions are found in the structure, one close to the reactive Cys122, and the other on a hydrophobic surface close to a symmetry-related molecule. There are major structural differences at the N-terminus of YhaK compared with similar structures that also display the bicupin fold (YhhW and hPirin). YhaK showed no quercetinase and peroxidase activity. However, reduced YhaK was very sensitive to reactive oxygen species (ROS). The complete, functional E. coli glutaredoxin or thioredoxin systems protected YhaK from oxidation. E. coli thioredoxin reductase and NADPH produced ROS and caused oxidation and oligomerization of reduced YhaK. Taken together, we propose that YhaK is the first of a new sub-class of bicupins that lack the canonical cupin metal-binding residues of pirins and may be involved in chloride binding and/or sensing of oxidative stress in enterobacteria.     

Delineation of the toxin coding fragments and an insect-specificity region of a dual toxicity Bacillus thuringiensis crystal protein gene

A series of deletion mutants have been constructed from the dual toxicity Bacillus thuringiensis aizawai IC1 (Bta IC1) crystal protein gene. The mutant toxin genes were expressed in Escherichia coli, their protein products purified and the authenticity of these mutant proteins confirmed immunologically. Analysis of the toxicity spectra of these mutants revealed that lepidopteran toxicity is located on the N-terminal region of the toxin between residues Ile30-Glu595. 3' deletion of a further 37 residues from Glu595 of the lepidopteran-specific toxin abolished lepidopteran toxicity but the resulting protein consisting of residues Ile30-Gly558 was still fully toxic to dipteran larvae and cells. Another mutant crystal protein gene truncated to encode residues between Ile30-Gly563 was toxic only to diptera. These data indicate that the determinants of lepidopteran specificity in the Bta IC1 toxin are located between residues Gly558-Glu595 and that the N-terminal portion of the toxin between Ile30-Gly558 is sufficient to express dipteran toxicity.     

Technical improvement to prevent DNA degradation of Leptospira spp. in pulsed field gel electrophoresis

Leptospirosis is a public health problem. Infection with pathogenic Leptospira occurs by exposure to many environments and is traditionally associated with occupational risk activities. Pulsed-field gel electrophoresis was used to investigate the epidemiological relatedness among Leptospira isolates. However, analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. This degradation can be significantly reduced by the inclusion of thiourea in the electrophoresis buffer, improving the analysis of DNA banding patterns.     

Crystal structure of a DNA binding protein from the hyperthermophilic euryarchaeon Methanococcus jannaschii

The Sac10b family consists of a group of highly conserved DNA binding proteins from both the euryarchaeotal and the crenarchaeotal branches of Archaea. The proteins have been suggested to play an architectural role in the chromosomal organization in these organisms. Previous studies have mainly focused on the Sac10b proteins from the crenarchaeota. Here, we report the 2.0 A resolution crystal structure of Mja10b from the euryarchaeon Methanococcus jannaschii. The model of Mja10b has been refined to an R-factor of 20.9%. The crystal structure of an Mja10b monomer reveals an alpha/beta structure of four beta-strands and two alpha-helices, and Mja10b assembles into a dimer via an extensive hydrophobic interface. Mja10b has a similar topology to that of its crenarchaeota counterpart Sso10b (also known as Alba). Structural comparison between the two proteins suggests that structural features such as hydrophobic inner core, acetylation sites, dimer interface, and DNA binding surface are conserved among Sac10b proteins. Structural differences between the two proteins were found in the loops. To understand the structural basis for the thermostability of Mja10b, the Mja10b structure was compared to other proteins with similar topology. Our data suggest that extensive ion-pair networks, optimized accessible surface area and the dimerization via hydrophobic interactions may contribute to the enhanced thermostability of Mja10b.     

Expression of the insecticidal crystal protein gene from a Gram-positive Bacillus thuringiensis in a Gram-negative Pseudomonas fluorescens mediated by protoplast fusion

Abstract Protoplast fusion between a Gram-negative strain Pseudomonas fluorescens having plant growth promoting activities and a Gram-positive Bacillus thuringiensis var. kurstaki HD 73 possessing insecticidal activity, was carried out to generate P. fluorescens hybrids possessing insecticidal activity. The antibiotic resistance markers of P. fluorescens (rif^r, nal^r) and the immunoreactivity to the antiserum raised against the crystal proteins of B. thuringiensis var. galleriae were used as selection markers for the hybrids. The hybrids exhibited lethal but differential activity in Heliothis armigera and in Spodoptera litura when compared to the parenthal B. thuringiensis strain. The anti-feedant activity which is characteristic of B. thuringiensis toxin was not observed in the hybrids. Although the presence of sequences homologous to the cloned insecticidal gene of B. thuringiensis was demonstrated, the Western blot analysis of cell extract of the hybrid (PK 105) showed that only low molecular mass crystal proteins (less than 40 kDa) could be detected under denaturing conditions. It indicates that the high molecular mass toxin peptide may be degraded by proteolysis. Besides this, a clear separation of lethal and anti-feedant activity of the B. thuringiensis toxin has been observed by this study.     

The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites

Periplasmic binding proteins (PBPs) comprise a protein superfamily that is involved in prokaryotic solute transport and chemotaxis. These proteins have been used to engineer reagentless biosensors to detect natural or non-natural ligands. There is considerable interest in obtaining very stable members of this superfamily from thermophilic bacteria to use as robust engineerable parts in biosensor development. Analysis of the recently determined genome sequence of Thermus thermophilus revealed the presence of more than 30 putative PBPs in this thermophile. One of these is annotated as a glucose binding protein (GBP) based on its genetic linkage to genes that are homologous to an ATP-binding cassette glucose transport system, although the PBP sequence is homologous to periplasmic maltose binding proteins (MBPs). Here we present the cloning, over-expression, characterization of cognate ligands, and determination of the X-ray crystal structure of this gene product. We find that it is a very stable (apo-protein Tm value is 100(+/- 2) degrees C; complexes 106(+/- 3) degrees C and 111(+/- 1) degrees C for glucose and galactose, respectively) glucose (Kd value is 0.08(+/- 0.03) microM) and galactose (Kd value is 0.94(+/- 0.04) microM) binding protein. Determination of the X-ray crystal structure revealed that this T. thermophilus glucose binding protein (ttGBP) is structurally homologous to MBPs rather than other GBPs. The di or tri-saccharide ligands in MBPs are accommodated in long relatively shallow grooves. In the ttGBP binding site, this groove is partially filled by two loops and an alpha-helix, which create a buried binding site that allows binding of only monosaccharides. Comparison of ttGBP and MBP provides a clear example of structural adaptations by which the size of ligand binding sites can be controlled in the PBP super family.     

Expression and characterization of a recombinant Cry1Ac crystal protein with enhanced green fluorescent protein in acrystalliferous Bacillus thuringiensis

AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined. METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae. CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.     

Construction, crystal structure and application of a recombinant protein that lacks the collagen-like region of BclA from Bacillus anthracis spores

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called BclA, which comprises a central collagen-like region (CLR) and a globular C-terminal domain. Here, the entire CLR coding sequence of BclA was removed, and the resulting protein (tBclA) produced in Escherichia coli. The crystallographic structure of tBclA was determined to 1.35 A resolution, and consists of an all-beta structure with a TNF-like jelly fold topology (12 beta-strands which form 2 beta-sheets of five strands each) consistent with previous studies on wild-type BclA. These globular domains are tightly packed into trimeric structures (surface shape complementarity; S (c) = 0.83), demonstrating that formation of the core structure of BclA is independent of the anchoring collagen-like region. A polyclonal antibody raised against tBclA recognized B. anthracis spores directly, and showed little cross-reactivity (<10%) with the spores of the closely related species Bacillus cereus and Bacillus thuringiensis, when compared to two other polyclonal antibodies raised against B. anthracis spore extracts and inactivated spores. The tBclA protein was used to purify a pool of specific antibodies from bovine colostrum whey samples from cows inoculated with the Sterne strain anthrax vaccine, which also showed reactivity with B. anthracis spores. Together, these results demonstrate that tBclA provides a safer and more effective way to the production and purification of antibodies with high binding affinity for B. anthracis spores. Biotechnol. Bioeng. 2008;99: 774-782. (c) 2007 Wiley Periodicals, Inc.     

The FI gene product of bacterial virus Lambda is related to theE. coli chromosomal protein NS2 and is involved in intracellular DNA organization

A likely function of the Lambda FI gene product (gpFI) is condensation of developmental forms of the bacteriophage DNA in the host cell. Several characteristics of gpFI support this hypothesis: it is similar in its structure and properties toE. coli NS proteins whose involvement in the bacterial DNA condensation has been established and it comigrates with DNA during fractionation of host cell lysate through a sucrose gradient.