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: 3.67). 10/2008; 74(22):6997-7001. DOI: 10.1128/AEM.01346-08
Source: PubMed


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
    • "In addition, cry genes can reside in small plasmids as well, such as pBMBt1 from B. thuringiensis subsp. darmstadiensis (6700 pb) (Loeza-Lara et al., 2005) or pBMB0228 from B. thuringiensis strain YBT-1518 (17,706 pb) (Guo et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Bacillus pumilus 15.1 strain, a recently described entomopathogenic strain active against Ceratitis capitata, contains at least two extrachromosomal elements, pBp15.1S and pBp15.1B. Given that B. pumilus is not a typical entomopathogenic bacterium, the acquisition of this extrachromosomal DNA may explain why B. pumilus 15.1 is toxic to an insect. One of the plasmids present in the strain, the pBp15.1S plasmid, was sub-cloned, sequenced and analyzed using bioinformatics to identify any potential virulence factor. The pBp15.1S plasmid was found to be 7785bp in size with a GC content of 35.7% and 11 putative ORFs. A replication module typical of small rolling circle plasmid and a sensing and regulatory system specific for plasmids was found in pBp15.1S. Additionally, we demonstrated the existence of ssDNA in plasmid preparations suggesting that pBp15.1S replicates by the small rolling circle mechanism. A gene cluster present in plasmid pPZZ84 from a distantly isolated B. pumilus strain was also present in pBp15.1S. The plasmid copy number of pBp15.1S in exponentially growing B. pumilus cells was determined to be 33 copies per chromosome. After an extensive plasmid characterization, no known virulence factor was found so a search in the other extrachromosomal elements of the bacteria is needed.
    Plasmid 09/2015; 82. DOI:10.1016/j.plasmid.2015.09.001 · 1.58 Impact Factor
  • Source
    • "The pHS18-4 plasmid harbors one insecticidal crystal gene, named cry56Aa, and the pHS18-9 plasmid harbors a cry54Ba gene. Most cry genes are located on large plasmids, except two reported genes, cry6Aa2 and cry55Aa1, which are located on the pBMBt1 and pBMB0288 plasmids (6.7 and 17.7 kb), respectively (Schnepf et al., 1998; Loeza-Lara et al., 2005; Guo et al., 2008). Our study showed that cry54Ba is located on a small plasmid, pHS18-9, which has a length of 7.4 kb. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus thuringiensis is a spore-forming bacterium that is a type of insect pathogen used in the field of microbial insect control. B. thuringiensis HS18-1 has effective toxicity for Lepidoptera and Diptera insects. It contains different types of parasporal crystal genes, including cry4Cb1, cry50Aa1, cry69Ab1, cry30Ga, cry30Ea, cry70Aa, cry71Aa, cry72Aa, cry56Aa and cry54Ba. Here, we report the complete genome sequence of B. thuringiensis HS18-1, which contains one circular gapless chromosome and nine circular plasmids. Copyright © 2015. Published by Elsevier B.V.
    Journal of Biotechnology 08/2015; 10(1). DOI:10.1016/j.jbiotec.2015.08.017 · 2.87 Impact Factor
  • 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 · 2.94 Impact Factor
Show more