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.

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    ABSTRACT: Bacillus thuringiensis (BT) has been widely used as a biopesticide primarily for the control of insect pests, but some B. thuringiensis strains target specifically nematodes. However, nematicidal virulence factors of B. thuringiensis are poorly investigated. Here, we describe virulence factors of nematicidal B. thuringiensis DB27 using C. elegans as a model. We show that B. thuringiensis DB27 kills a number of free-living and animal-parasitic nematodes via intestinal damage. Its virulence factors are plasmid-encoded Cry protoxins, since plasmid-cured derivatives do not produce Cry proteins and are not toxic to nematodes. Whole genome sequence of B. thuringiensis DB27 revealed multiple potential nematicidal factors, including several Cry-like proteins encoded by different plasmids. Two of these proteins appear to be novel and show high similarity to Cry21Ba1. Named Cry21Fa1 and Cry21Ha1, they were expressed in E. coli and fed to C. elegans, resulting in intoxication, intestinal damage and death of nematodes. Interestingly, the effect of the two protoxins on C. elegans is synergistic (synergism factor 1.8-2.5). Using purified proteins, we determined the LC50 for Cry21Fa1 (13.6 μg/ml) and Cry21Ha1 (23.9 μg/ml), which is comparable to the LC50 of nematicidal Cry5B. Finally, we found that signaling pathways, which protect C. elegans against Cry5B toxin, are also required for protection against Cry21Fa1. Thus, B. thuringiensis DB27 produces novel nematicidal protoxins Cry21Fa1 and Cry21Ha1 with synergistic action, which highlights the importance of naturally isolated strains as a source of novel toxins.
    Applied and Environmental Microbiology 03/2014; · 3.95 Impact Factor
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    ABSTRACT: There has been considerable effort made in recent years for research groups and other organizations to build up large collections of strains of Bacillus thuringiensis in the search for genes encoding novel insecticidal toxins, or encoding novel metabolic pathways. Whilst next generation sequencing allows the detailed genetic characterization of a bacterial strain with relative ease it is still not practicable for large strain collections. In this work we assess the practicability of mining a mixture of genomic DNA from a two thousand strain collection for particular genes. Using PCR the collection was screened for both a rare (cry15) toxin gene as well as a more commonly found gene (vip3A). The method was successful in identifying both a cry15 gene and multiple examples of the vip3A gene family including a novel member of this family (vip3Aj). A number of variants of vip3Ag were cloned and expressed, and differences in toxicity observed despite extremely high sequence similarity.
    Journal of Invertebrate Pathology 08/2014; · 2.67 Impact Factor

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