[Show abstract][Hide abstract] ABSTRACT: Cyt1Aa is a major mosquitocidal protein synthesized during sporulation of Bacillus thuringiensis subsp. israelensis, composing more than 50 % of its parasporal body. This high level of synthesis is due to several factors including three strong sporulation-dependent promoters, a strong transcription termination sequence, and an associated 20-kDa helper protein. Cyt1Aa's toxicity is low compared to the Cry proteins of this species, namely, Cry4Aa, Cry4Ba, and Cry11Aa, but it nevertheless plays an important role in the biology of B. thuringiensis subsp. israelensis in that it synergizes their mosquitocidal toxicity and suppresses the evolution of resistance. In the present study, the effects of using different cyt1Aa promoter combinations and plasmid copy number on synthesis of Cyt1Aa were evaluated. Using the 4Q7 (plasmid-cured) strain of B. thuringiensis subsp. israelensis as an experimental host, a plasmid copy number of two or three yielded no Cyt1Aa, whereas a copy number of four yielded only small crystals, even when expression was driven by one of the wild-type promoters. However, using all three wild-type promoters and a plasmid copy number of 20 yielded Cyt1A crystals tenfold larger than those produced by one promoter and a plasmid copy number of four. High levels of Cyt1Aa synthesis resulted in significantly fewer spores per unit medium and imperfectly formed crystals. Similar results were obtained when Cyt1Aa synthesis was evaluated using the same expression constructs in a mutant strain of B. thuringiensis subsp. israelensis that lacks the cyt1Aa gene.
No preview · Article · Sep 2015 · Current Microbiology
[Show abstract][Hide abstract] ABSTRACT: The family Iridoviridae of the superfamily Megavirales currently consists of five genera. Three of these, Lymphocystivirus, Megalocytivirus and Ranavirus, are composed of species that infect vertebrates, and the other two, Chloriridovirus and Iridovirus, contain species that infect invertebrates. Until recently, the lack of genomic sequence data limited investigation of the evolutionary relationships between the invertebrate iridoviruses (IIVs) and vertebrate iridoviruses (VIVs), as well as the relationship of these viruses to those of the closely related family Ascoviridae, which only contains species that infect insects. To help clarify the phylogenetic relationships of these viruses, we recently published the annotated genome sequences of five additional IIV isolates. Here, using classical approaches of phylogeny via maximum likelihood, a Bayesian approach, and resolution of a core protein tree, we demonstrate that the invertebrate and vertebrate IV species constitute two lineages that diverged early during the evolution of the family Iridoviridae, before the emergence of the four IIV clades, previously referred to as Chloriridoviruses, Polyiridoviruses, Oligoiridoviruses and Crustaceoiridoviruses. In addition, we provide evidence that species of the family Ascoviridae have a more recent origin than most iridoviruses, emerging just before the differentiation between the Oligoiridoviruses and Crustaceoiridovirus clades. Our results also suggest that after emergence, based on their molecular clock, the ascoviruses evolved more quickly than their closest iridovirus relatives.
No preview · Article · Jan 2015 · Molecular Phylogenetics and Evolution
[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 fromthe 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.
Full-text · Article · Sep 2014 · Proceedings of the National Academy of Sciences
[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.
[Show abstract][Hide abstract] ABSTRACT: Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridoviruses 22 (IIV22) and 25 (IIV25) were originally isolated from a single sample of blackfly larva (Simulium spp., order Diptera) collected from the Ystwyth river near Aberystwyth, Wales. Recently, the genomes of IIV22 (197.7 kbp) and IIV25 (204.8 kbp) were sequenced and reported. Here, we describe the complete genome sequence of IIV22A, a variant that was isolated from the same pool of virions collected from the blackfly larva from which the IIV22 virion genome originated. The IIV22A genome, 196.5 kbp, is smaller than IIV22. Nevertheless, it contains 7 supplementary putative ORFs. Its analysis enables evaluation of the degree of genomic polymorphisms within an IIV isolate. Despite the occurrence of this IIV variant with IIV22 and IIV25 in a single blackfly larva and the features of their DNA polymerase, we found no evidence of lateral genetic transfers between the genomes of these two IIV species.
Full-text · Article · Jun 2014 · Standards in Genomic Sciences
[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.
No preview · Article · May 2014 · Systematic and Applied Microbiology
[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.
Full-text · Article · Apr 2014 · Journal of General Virology
[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.
No preview · Article · Jan 2014 · Journal of Invertebrate Pathology
[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.
No preview · Article · Nov 2013 · Archives of Virology
[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.
Full-text · Article · Oct 2013 · Journal of Invertebrate Pathology
[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.
No preview · Article · Sep 2013 · Journal of Asia-Pacific Entomology
[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.
Full-text · Article · Jun 2013 · Archives of Virology
[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.
Full-text · Article · Jun 2013 · Journal of General Virology
[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.
Full-text · Article · Jun 2013 · Journal of Microbiology and Biotechnology
[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 a 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 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 had estimated 50% lethal concentrations of 2.9 to 7.9 μg/ml; however, Cry30Ca with
or without ORF2 was not toxic to this mosquito.
Full-text · Article · Mar 2013 · Applied and Environmental Microbiology
[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.
No preview · Article · Feb 2013 · Molecular Phylogenetics and Evolution
[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.
Full-text · Article · Jan 2013 · Journal of Microbiology and Biotechnology
[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.
Full-text · Article · Nov 2012 · Transgenic Research