New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518

State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
Applied and Environmental Microbiology (Impact Factor: 3.95). 10/2008; 74(22):6997-7001. DOI: 10.1128/AEM.01346-08
Source: PubMed

ABSTRACT We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes--cry55Aa1, cry6Aa2, and cry5Ba2--were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

Download full-text


Available from: Peng Donghai, Oct 08, 2014
  • Source
    • "B. thuringiensis Cry and Cyt proteins are primarily toxic to a wide range of insect orders, but also to mites and protozoa [4,5]. Several families of nematicidal Cry proteins (Cry5, Cry6, Cry12, Cry13, Cry14, Cry21, Cry55) have also been described [16,17,18]. However, other nematicidal virulence factors of B. thuringiensis and the spectrum of their activity are poorly investigated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence.
    Toxins 07/2014; 6(7):2050-2063. DOI:10.3390/toxins6072050 · 2.48 Impact Factor
  • Source
    • "Meanwhile, biologically active crystal B. thuringiensis proteins also were found to be toxic against various nematode species, and each tested nematode species was susceptible to at least one toxin (Mozgovaya et al. 2002, Wei et al. 2003). The genes cry55Aa1, cry5Ba2, and cry6Aa2 were found in B. thuringiensis YBT-1518 and their proteins were toxic to the root-knot nematode Meloidogyne hapla (Guo et al. 2008, Zhang et al. 2012), while cry6Aa2 was detected to be negatively regulated by orf2, which was coexpressed with cry6Aa2 (Yu et al. 2008). The pore-forming protein Cry5B was also reported to elicit the pathogenicity of Bacillus sp. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In addition to being used increasingly as a model system in modern molecular biology studies, the free-living nematode Caenorhabditis elegans (Maupas, 1900) is an important pathogen in fungi and straw mushrooms. In this study, Bacillus thuringiensis strain 010 was found to have significantly detrimental activity against C. elegans. To further characterize this activity, the toxicological mechanism was elucidated at molecular level. Genes encoding for crystal protein and chitinase were isolated, cloned, and sequenced. However, the toxicity was detected only in the chitinase. Under transmission electron microscopy, change in the body wall and gut structures of C. elegans was observed, and thus degeneration of body wall and gut in the worms was also investigated. Further bioassay also confirmed the mortality of C. elegans fed with Escherichia coli TB1 strain. These observations suggest great potential for B. thuringiensis 010 as a biocontrol agent against C. elegans and other nematodes.
    Journal of Economic Entomology 04/2014; 107(2):551--8. DOI:10.1603/EC13201 · 1.61 Impact Factor
  • Source
    • "Several families of crystal proteins have been observed toxic to nematode, such as Cry5B, Cry6A, Cry14A, Cry21A and Cry55A. (Wei et al. 2003; Guo et al. 2008). Among the documented nematicidal crystal proteins, Cry5B and Cry6A represent two distinct groups (Marroquin et al. 2000; Wei et al. 2003), and all can inhibit infection of root-knot nematode to tomato (Li et al. 2007, 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The group of nematicidal crystal protein Cry6A shares very low identity and exhibits different structure with Cry5B, another well-studied group of nematicidal crystal protein produced by Bacillus thuringiensis. In this study, we assayed the susceptibility of bre mutants (Caenorhabditis elegans with resistance to Cry5B) to Cry6Aa2, and examined the synergistic activity between Cry6Aa2 and Cry5Ba2. Our results show that all bre mutants are susceptible to Cry6Aa2 on the lethal activity, growth inhibition, fertility, and exhibit no cross-resistance to Cry6Aa2. Moreover, all combinations of Cry6Aa2 and Cry5Ba2 with serial ratios exhibit significant synergism to C. elegans, and the highest synergistic effect was observed when Cry6Aa2 and Cry5Ba2 were mixed with a ration of 4:1. The susceptibility of bre mutants to Cry6A and synergistic activity between Cry6A and Cry5B may be attributed to the diverse action mode, because of different structure of the two nematicidal crystal protein toxins. This article is protected by copyright. All rights reserved.
    Letters in Applied Microbiology 01/2014; 58(6). DOI:10.1111/lam.12219 · 1.75 Impact Factor
Show more