A pair of adjacent genes, cry5Ad and orf2-5Ad , encode the typical N- and C-terminal regions of a Cry5Aδ-endotoxin as two separate proteins in Bacillus thuringiensis strain L366

CSIRO Livestock Industries, Queensland Bioscience Precinct, St Lucia, Qld, Australia.
FEMS Microbiology Letters (Impact Factor: 2.12). 02/2008; 278(1):115-20. DOI: 10.1111/j.1574-6968.2007.00987.x
Source: PubMed


A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.

Download full-text


Available from: Peter Josh, Oct 17, 2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Published data on insecticidal activity of crystal proteins from Bacillus thuringiensis are incorporated into the Bt toxin specificity relational database. To date, 125 of the 174 holotype known toxins have been tested in approximately 1700 bioassays against 163 test species; 49 toxins have not been tested at all; 59 were tested against 71 Lepidoptera species in 1182 bioassays; 53 toxins were tested against 23 Diptera species in 233 bioassays; and 47 were tested against 39 Coleoptera species in 190 bioassays. Activity spectra of the tested toxins were summarized for each order. Comparisons of LC(50) values are confounded by high variability of the estimates, mostly due to within-species variation in susceptibility, and errors associated with estimation of toxin protein content. Limited analyses suggest that crystal protein toxicity is not affected by quarternary toxin rank or host used for gene expression, but that pre-ingestion treatment by solubilization or enzymatic processing has a large effect. There is an increasing number of toxin families with cross-order activity, as 15 of the 87 families (secondary rank) that are pesticidal are active against more than one order. Cross-order activity does not threaten environmental safety of B. thuringiensis-based pest control because toxins tend to be much less toxic to taxa outside the family's primary specificity range.
    No preview · Article · Apr 2009 · Journal of Invertebrate Pathology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Insect cadherin proteins localized in the midgut epithelium were identified as receptors for Bacillus thuringiensis insecticidal crystal proteins (Cry toxins). These cadherins facilitated toxin monomer oligomerization and mediated oligomer binding to secondary receptors. It has been reported that Manduca sexta, Helicoverpa armigera, Anopheles gambiae and Diabrotica virgifera cadherin toxin binding regions function as synergists for Cry1A, Cry4Ba and Cry3A toxicity against target insects. In the present study, the toxin binding region fragment of the H. armigera cadherin (hacad1) gene was cloned and fused with the promoter of the cry3Aa gene. The fusion gene pro3Aa-hacad1 and the cry1Ac gene were inserted into shuttle vector pHT304 and introduced into B. thuringiensis acrystalliferous strain BMB171 for coexpression (resulting in recombinant strain BMB1073). SDS-PAGE and mass spectrum analysis showed that BMB1073 could express HaCad1 and Cry1Ac proteins together. Bioassay results demonstrated that insecticidal activities against H. armigera and Spodoptera exigua could be increased 5.1-fold and 6.5-fold, respectively, by BMB1073 compared with the strain which can only express the Cry1Ac protein. Our discovery showed that coexpression of HaCad1 and Cry1Ac toxin in B. thuringiensis enhanced the insecticidal activity of Cry1Ac toward Lepidoptera insects. This finding also revealed a novel strategy for engineering strains and transgenic plants with higher insecticidal activity.
    Full-text · Article · May 2010 · Research in Microbiology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus thuringiensis, which is well known as an entomopathogen, has been accepted by the public as a safe bioinsecticide. The natural ecology of this bacterium has never been particularly clear, with views ranging from it being an obligate pathogen to an opportunist pathogen that can otherwise exist as a soil saprophyte or a plant endophyte. This confusion has recently led to it being considered as an environmental pathogen that has evolved to occupy a diverse set of environmental niches in which it can thrive without needing a host. A significant driving force behind this classification is the fact that B. thuringiensis is found in high numbers in environments that are not occupied by the insect hosts to which it is pathogenic. It is our opinion that the ubiquitous presence of this bacterium in the environment is the result of a variety of vectoring systems, particularly those that include nematodes. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Mar 2015 · Trends in Microbiology