[Show abstract][Hide abstract] ABSTRACT: It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.
Proceedings of the National Academy of Sciences of the United States of America. 08/2014;
[Show abstract][Hide abstract] ABSTRACT: The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation 'Candidatus Rickettsiella isopodi' is introduced for this new lineage of isopod-associated Rickettsiella bacteria.
Systematic and Applied Microbiology 05/2014; · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 31 (IIV31) was originally isolated from pill bug adults of Armadillidium vulgare (class Crustacea, order Isopoda, sub-order Oniscidea) found in Southern California on the campus of the University of California, Riverside. IIV31 virions are icosahedral, have a diameter of about 135 nm, and contain a dsDNA genome of 220.22 kbp in length, 35.09 % GC content and 203 open reading frames. Here, we describe the complete genome sequence of this virus and its annotation. This is the eighth genome sequence of an invertebrate iridovirus reported.
Journal of General Virology 04/2014; · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 30 (IIV30) was originally isolated from a larva of the corn earworm, Helicoverpa zea (order Lepidoptera, Family Noctuidae) in western Australia. The IIV30 virions are icosahedral, have a diameter of about 130 nm, and contain a dsDNA genome of 198.5 kbp with 28.11% in GC content and 177 coding sequences. Here we describe its complete genome sequence and annotate the genes for which we could assign a putative function. This is the sixth genome sequence of an invertebrate iridovirus reported.
Journal of Invertebrate Pathology 01/2014; · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. Invertebrate iridovirus 25 (IIV-25) was originally isolated from the larva of a blackfly (Simulium spp., order Diptera) found in the Ystwyth river near Aberystwyth, Wales. IIV-25 virions are icosahedral, have a diameter of ~130 nm, and contain a dsDNA genome of 204.8 kbp, with a G+C content of 30.32 %, that codes for 177 proteins. Here, we describe the complete genome sequence of this virus and its annotation. This is the fifth genome sequence of an invertebrate iridovirus reported.
Archives of Virology 11/2013; · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interaction of Mtx toxins from Lysinibacillus sphaericus (formerly Bacillus sphaericus) with Bacillus thuringiensis subsp. israelensis Cry toxins and the influence of such interactions on Cry-resistance were evaluated in susceptible and Cry-resistant Culex quinquefasciatus larvae. Mtx-1 and Mtx-2 were observed to be active against both susceptible and resistant mosquitoes; however varying levels of cross-resistance toward Mtx toxins were observed in the resistant mosquitoes. A 1:1 mixture of either Mtx-1 or Mtx-2 with different Cry toxins generally showed moderate synergism, but some combinations were highly toxic to resistant larvae and suppressed resistance. Toxin synergy has been demonstrated to be a powerful tool for enhancing activity and managing Cry-resistance in mosquitoes, thus Mtx toxins may be useful as components of engineered bacterial larvicides.
Journal of Invertebrate Pathology 10/2013; · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Four mRNA variants in pooled cDNA samples of the glassy-winged sharpshooter, Homalodisca coagulata, encoding a putative regulator of cation transporters were identified. RT-PCR showed that the hc-chaC variants were expressed during different stages of insect development and in different tissues and sexes. Structural analysis of the hc-chaC gene indicated that intron and exon sequences of the mRNA variants were identical, and similar to chaC-like genes of other insects. The Hc-ChaC protein (22.5 kDa) contained the conserved FGYGSL K+-binding motif near its amino-terminus, and the carboxy-terminal region contained two coiled-coil motifs, which had similarity to the PDZ domain present in well-characterized Na+/H+ exchanger regulatory factors. Our analyses suggest that Hc-ChaC is a regulator of K+ transporters such as K+/H+ antiporters.
Journal of Asia-Pacific Entomology 09/2013; 16(3):301–307. · 0.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nucleocytoplasmic large DNA viruses (NCLDVs) comprise a monophyletic group of viruses that infect animals and diverse unicellular eukaryotes. The NCLDV group includes the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Mimiviridae and the proposed family "Marseilleviridae". The family Mimiviridae includes the largest known viruses, with genomes in excess of one megabase, whereas the genome size in the other NCLDV families varies from 100 to 400 kilobase pairs. Most of the NCLDVs replicate in the cytoplasm of infected cells, within so-called virus factories. The NCLDVs share a common ancient origin, as demonstrated by evolutionary reconstructions that trace approximately 50 genes encoding key proteins involved in viral replication and virion formation to the last common ancestor of all these viruses. Taken together, these characteristics lead us to propose assigning an official taxonomic rank to the NCLDVs as the order "Megavirales", in reference to the large size of the virions and genomes of these viruses.
Archives of Virology 06/2013; · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 22 (IIV22) was originally isolated from the larva of a blackfly (Simulium spp., order Diptera) found in the Ystwyth river, near Aberystwyth, Wales. IIV22 virions are icosahedral, have a diameter of about 130 nm, and contain a dsDNA genome that is 197.7 kbp in length, 28.05 % in GC content and contains 167 coding sequences. Here, we describe the complete genome sequence of this virus and its annotation. This is the fourth genome sequence of an invertebrate iridovirus reported.
Journal of General Virology 06/2013; · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Cyt1Aa protein of Bacillus thuringiensis susbp. israelensis elaborates demonstrable toxicity to mosquito larvae, but more importantly enhances larvicidal activity of this species Cry proteins (Cry11Aa, Cry4Aa and Cry4Ba) and delays the phenotypic expression of resistance to these that has evolved in Culex quinquefasciatus. It is also known that Cyt1Aa, which is highly lipophilic, synergizes Cry11Aa by functioning as a surrogate membrane-bound receptor for the latter protein. Little is known, however, about whether Cyt1Aa can interact similarly with other Cry proteins not primarily mosquitocidal, for example, Cry2Aa, which is active against lepidopteran larvae, but essentially inactive or has very low toxicity to mosquito larvae. Here we demonstrated by ligand binding and enzyme-linked immunosorbent assays that these toxins form intermolecular complexes in vitro, and in addition showed that Cyt1Aa facilitates binding of Cry2Aa throughout the midgut of C. quinquefasciatus larvae. As Cry2Aa and Cry11Aa share structural similarity in domain II, it appears that the interaction between these toxins could results from a similar mechanism previously proposed for Cry11Aa and Cyt1Aa. Finally, despite the observed interaction between Cry2Aa and Cyt1Aa, only a 2-fold enhancement in toxicity resulted against C. quinquefasciatus. Regardless, our results suggest that Cry2Aa could be a useful component of mosquitocidal endotoxin complements being developed for recombinant strains of B. thuringiensis subsp. israelensis and B. sphaericus aimed at improving efficacy of commercial products and avoiding resistance.
Journal of Microbiology and Biotechnology 06/2013; · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The total protoxin complement in the parasporal body of mosquitocidal strain, Bacillus thuringiensis subsp. jegathesan 367, was determined by use of polyacrylamide gel block coupled to mass spectrometry. A total of eight protoxins were identified from this strain, including five reported protoxins (Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa, and Cyt2Bb) as well as three previously undescribed (Cry30Ca, Cry60Aa, and Cry60Ba) in this isolate. It was interesting to note that the encoding genes of three new protoxins existed as cry30Ca-gap-orf2 and cry60Ba-gap-cry60Aa. The cry30Ca and a downstream orf2 gene were oriented in the same direction and separated by 114 bp, and cry60Ba was located 156 bp upstream from and in the same orientation to cry60Aa. The three new protoxin genes were cloned from B. thuringiensis subsp. jegathesan and expressed in an acrystalliferous strain under the control of cyt1A gene promoters and the STAB-SD stabilizer sequence. Recombinant strain containing only cry30Ca did not produce visible inclusion under microscope observation, while that containing both cry30Ca and orf2 could produce large inclusions. Cry60Aa and Cry60Ba synthesized either alone or together in the acrystalliferous host could yield large inclusions. In bioassays using the fourth instar larvae of Culex quinquefasciatus, Cry60Aa and Cry60Ba alone or together has estimated 50% lethal concentrations of 2.9 - 7.9 μg/ml; however, Cry30Ca with or without ORF2 were not toxic to this mosquito.
Applied and environmental microbiology 03/2013; · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inteins are self-splicing proteins that occur in-frame within host-coded proteins. DNA elements coding for inteins insert specifically in highly conserved motifs of target genes. These mobile genetic elements have an uneven distribution, and thus far have been found only in certain species of bacteria, archaea and fungi, a few viruses of algae and amoebozoa and in the entomopathogen, Chilo iridescent virus (CIV). Here we report the discovery of seven new inteins parasitizing iridoviruses infecting metazoans: three within their δ DNA polymerase genes, and four in genes coding for their large ribonucleotide reductase subunit. Analyses of coding sequences suggests that these inteins were acquired by ancestors shared by viruses currently classified as members of different families of viruses with large double-stranded (ds) DNA genomes, and then were maintained by vertical transmission, or lost. Of significant interest is the finding that inteins present in the δ DNA polymerases of iridoviruses insert at a different location into the YGDTDS motif when compared to those found in other viruses and prokaryotes. In addition, our phylogenetic investigations suggest that inteins present in the δ DNA polymerases of these viruses might have an origin different from those found in prokaryotes. Finally, we use the sequence features of the intein insertion sites in host genes to discuss the high polymorphisms of inteins within and among viral species and the immunity of their genetic counterparts in the eukaryotic hosts of these viruses.
Molecular Phylogenetics and Evolution 02/2013; · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Cyt1Aa protein of Bacillus thuringiensis subsp. israelensis is known to synergize mosquitocidal proteins of B. thuringiensis and Bacillus sphaericus strains. Cyt1Aa is highly lipophilic, and after binding in vivo to the midgut microvillar membrane serves as a "receptor" for mosquitocidal Cry proteins, which subsequently form cation channels that kill mosquito larvae. Here we report that Cyt1Aa can serve a similar function for lepidopteran-specific Cry proteins of B. thuringiensis in certain mosquito larvae. Engineering Cyt1Aa into the HD-1 isolate of B. thuringiensis subsp. kurstaki enhanced toxicity against 4th instars of Aedes aegypti, but not against 4th instars of Culex quinquefasciatus.
Journal of Microbiology and Biotechnology 01/2013; 23(1):88-91. · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mendelian crosses were used to study the mode of inheritance of Cry toxin resistance in a Culex quinquefasciatus Say (Diptera: Culicidae) colony (CqAB11A) that evolved insecticide resistance under laboratory selection with a deletion mutant of Bacillus thuringiensis subsp. israelensis de Barjac lacking the Cyt1Aa toxin component but containing its three major Cry toxins, Cry4Aa, Cry4Ba, and Cry11Aa. High levels of resistance were observed to Cry toxins. F1 offspring of reciprocal crosses to a sensitive colony showed intermediate levels of resistance with no maternal effect, indicating autosomal inheritance. Dose-response data for backcross offspring deviated significantly from the monofactorial model when tested with Cry4Aa + Cry4Ba + Cry11Aa, Cry4Aa + Cry4Ba, or Cry11Aa. However, tests with Cry11Ba from B. thuringiensis subsp. jegathesan (Seleena, Lee, Lecadet) fit the monofactorial model. Dominance of F1 offspring was calculated at different concentrations of Cry-toxin suspensions and, as reported for other Cry-resistant Culex, generally decreased as concentration increased. A subset of colony CqAB11A was reared without selection pressure for 18 generations with little change in susceptibility, indicating a highly homozygous population. Consistent with reports for other Cry-resistant Culex, the data show these mosquitoes evolved resistance to B. thuringiensis Cry toxins at multiple loci in response to selection pressure and that cross-resistance to Cry11Ba was conferred by one of those loci.
Journal of Medical Entomology 07/2012; 49(4):886-94. · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC(95)] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC(95) = 8.2 μg/ml).
Applied and environmental microbiology 03/2012; 78(6):2005-12. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in B. thuringiensis subsp. israelensis, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.
Journal of bacteriology 12/2011; 194(6):1562-71. · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Environmental risk assessments (ERA) support regulatory decisions for the commercial cultivation of genetically modified (GM) crops. The ERA for terrestrial agroecosystems is well-developed, whereas guidance for ERA of GM crops in aquatic ecosystems is not as well-defined. The purpose of this document is to demonstrate how comprehensive problem formulation can be used to develop a conceptual model and to identify potential exposure pathways, using Bacillus thuringiensis (Bt) maize as a case study. Within problem formulation, the insecticidal trait, the crop, the receiving environment, and protection goals were characterized, and a conceptual model was developed to identify routes through which aquatic organisms may be exposed to insecticidal proteins in maize tissue. Following a tiered approach for exposure assessment, worst-case exposures were estimated using standardized models, and factors mitigating exposure were described. Based on exposure estimates, shredders were identified as the functional group most likely to be exposed to insecticidal proteins. However, even using worst-case assumptions, the exposure of shredders to Bt maize was low and studies supporting the current risk assessments were deemed adequate. Determining if early tier toxicity studies are necessary to inform the risk assessment for a specific GM crop should be done on a case by case basis, and should be guided by thorough problem formulation and exposure assessment. The processes used to develop the Bt maize case study are intended to serve as a model for performing risk assessments on future traits and crops.
Transgenic Research 11/2011; 21(4):813-42. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine whether the 20-kDa chaperone-like protein of Bacillus thuringiensis ssp. israelensis enhances synthesis, crystallization and solubility of the Cry3A coleopteran toxin and whether the crystalline inclusions produced are toxic to neonates of the Colorado potato beetle, Leptinotarsa decemlineata.
The cry3A gene was expressed in the 4Q7 strain of B. thuringiensis ssp. israelensis in the absence or presence of the 20-kDa gene. The 20-kDa protein enhanced Cry3A yield by 2·7-fold per unit of fermentation medium. Crystal volumes averaged 2·123 and 0·964 μm(3) when synthesized in, respectively, the presence or absence of the 20-kDa protein. Both crystals were soluble at pH 5 and pH 6; however, the larger crystal was 1·7× and 1·5× more soluble at, respectively, pH 7 and pH 10. No significant difference in toxicity against L. decemlineata neonates was observed.
This report demonstrated that the 20-kDa chaperone-like protein enhances yield, volume and solubility of the coleopteran Cry3A crystalline inclusions per unit crystal/spore mixture.
This is the first report showing that an accessory protein (20-kDa) could enhance synthesis and crystallization of Cry3A, a finding that could be beneficial for commercial production of this coleopteran-specific insecticidal protein for microbial insecticides and possibly even for transgenic crops.
Letters in Applied Microbiology 11/2011; 54(2):88-95. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Larvae of the potato tubermoth (PTM), Phthorimaea operculella, feed on potato plants and tubers and are a major pest in the tropics and subtropics worldwide, causing up to 100% damage. The PTM granulovirus (PhopGV) provides significant potato protection, but little is known about its effect on larval development or its histopathology. Here we show that only 10% of larvae exited from PhopGV-treated tubers (1.4×10(8) granule/ml), lagging significantly behind controls, and most of these died by 72 h after emergence. Histopathology studies showed the fat body and epidermis were the principal tissues infected. PhopGV morphogenesis was similar to other GVs, the exception being small vesicles between mature granules.
Journal of Invertebrate Pathology 06/2011; 108(1):52-5. · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The insecticidal and psychrotropic potential of 132 new isolates of Bacillus thuringiensis from northeastern Poland (74 from animals and 58 from soil) were determined by screening these for vip and cry genes encoding, respectively, vegetative insecticidal proteins (Vip) and Cry proteins, and cspA that encoded the CspA cold shock protein that confers psychrotropy in Bacillus species. The vip3A gene, encoding Vip3A toxic to lepidopterans, was found in ~5% of the isolates from animals and ~17% the isolates from soil, whereas coleopteran-specific vip1 and vip2 genes were present in 8% of the isolates from soil. Nucleotide sequences of vip3A-specific amplicons were highly conserved, with only a few containing minor differences from vip3A. Despite the high level of vip3A conservation, isolates harbouring the gene demonstrated a high level of heterogeneity based on whole-cell genomic DNA RFLP analysis with pulsed-field gel electrophoresis (PFGE) and plasmid profiling. Eight isolates positive for vip3A contained cry1 and six also harboured the cry2 gene, which encodes an endotoxin toxic to lepidopteran insects. However, none of these isolates contained cry genes coding for proteins toxic to coleopteran or dipteran insects. Due to the known potential for synergistic interactions between Vip and Cry proteins, the isolates positive for vip3A and cry genes may be used in resistance management strategies directed against lepidopteran larvae. Finally, all of the B. thuringiensisvip3A-positive isolates harboured the cspA gene, but only two were confirmed to be psychrotrophs.
Biocontrol Science and Technology 04/2011; 21(4):461-473. · 0.71 Impact Factor