Cloning and Characterization of Two Novel Crystal Protein Genes, cry54Aa1 and cry30Fa1, from Bacillus thuringiensis Strain BtMC28

Tan, Furong; Zhu, Jun; Tang, Jie; Tang, Xueming; Wang, Yunlong; Zheng, Anqi; Li, Ping

doi:10.1007/s00284-009-9386-y

Cited by 28 publications

(18 citation statements)

References 17 publications

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“…The same approach to the design of universal primers to amplify cry genes has been reported before (6,13,26,28); however, most of the approaches are limited to amplification of specific groups, or experimental evidence to prove their universality by finding totally new groups within the cry family is lacking.…”

Section: Discussionmentioning

confidence: 99%

“…Techniques such as PCR-restriction fragment length polymorphism analysis and exclusive PCR (E-PCR) are based on the use of universal primers, although most of these primers are designed to recognize specific groups (i.e., cry1 genes), or at best, they recognize a few related groups (i.e., cry1, cry8, and cry9 genes) (13,26). Interestingly, both techniques may enhance the ability to identify known cry genes, if our proposed primer system is used.…”

Section: Discussionmentioning

confidence: 99%

“…The search for novel Cry toxins has followed different strategies, such as (i) the development of DNA libraries and their expression in acrystalliferous mutants of B. thuringiensis (24); (ii) hybridization with degenerate oligonucleotides, partial sequences, or complete cry genes used as probes (5,12); and (iii) PCR amplification of putative novel cry genes by using multiple primers (14) or a combination of general primers, followed either by restriction analysis (26) or by a second amplification using a mixture of general and specific primers (13). These strategies have shown certain levels of efficiency in detecting new cry genes, but they also have some limitations.…”

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Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

Noguera

Ibarra

2010

Appl Environ Microbiol

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On the basis of the known cry gene sequences of Bacillus thuringiensis, three sets of primers were designed from four conserved blocks found in the delta-endotoxin-coding region. The primer pairs designed amplify the regions between blocks 1 and 5, 2 and 5, and 1 and 4. In silico analyses indicated that 100% of the known three-domain cry gene sequences can be amplified by these sets of primers. To test their ability to amplify known and unknown cry gene sequences, 27 strains from the CINVESTAV (LBIT series) collection showing atypical crystal morphology were selected. Their DNA was used as the template with the new primer system, and after a systematic amplification and sequencing of the amplicons, each strain showed one or more cry-related sequences, totaling 54 different sequences harbored by the 27 strains. Seven sequences were selected on the basis of their low level of identity to the known cry sequences, and once cloning and sequencing of the complete open reading frames were done, three new cry-type genes (primary ranks) were identified and the toxins that they encode were designated Cry57Aa1, Cry58Aa1, and Cry59Aa1 by the B. thuringiensis Toxin Nomenclature Committee. The rest of the seven sequences were classified Cry8Ka2, Cry8-like, Cry20Ba1, and Cry1Ma1 by the committee. The crystal morphology of the selected strains and analysis of the new Cry protein sequences showed interesting peculiarities.Bacillus thuringiensis has been safely used for the control of insect pests within the orders Lepidoptera, Coleoptera, and Diptera for the last 50 years (19). It is an aerobic, Grampositive bacterium whose insecticidal activity is based on the presence of parasporal crystalline inclusions formed during the sporulation process. These parasporal bodies or crystals are assembled by the so-called Cry proteins, expressed by the cry genes (21).B. thuringiensis shows great variability, as has been demonstrated by the huge number of strains isolated around the world (19), by the number of serotypes known to date (a total of 84) (20), and by the great number of different cry gene sequences accumulated so far (a total of 492) (8), as well as by the number of molecular characterization tools that have been developed, such as sequencing of the flagellin gene and of the gyrB and aroE genes, the band patterns from repetitive extragenic palindromic-PCR analyses, and the plasmid patterns, among others (17,18,25,27), all indicating the great variability within this species.In spite of this variability, some similarity has been found in the three-domain Cry proteins, evidenced by the presence of three conserved domains in the tertiary structure of the active toxin (delta-endotoxin), even from Cry toxins showing low levels of identity (i.e., the Cry1-type, Cry3-type, and Cry4-type toxins): one domain with a bundle of ␣ helices and two domains of ␤ sheets (3). This uniformity is, at least in part, a reflection of the five conserved blocks in the gene structure, which are present in almost all the cry genes (10).The great number of...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

Noguera

Ibarra

2010

Appl Environ Microbiol

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“…A given Cry protein has a fairly narrow range of target organisms against which it is effective. For example, Cry1-, Cry2-, and Cry9-type toxins are active against Lepidoptera; Cry3, Cry7, Cry8, Cry18, etc., are effective against Coleoptera; Cry2, Cry4, Cry10, Cry11, Cry16, etc., are effective against Diptera; and Cry5 and Cry6 toxins exhibit activity against plant-parasitic nematodes and plant-pathogenic nematodes (1,11,23,41,48).Locusts (Orthoptera) are pests that cause extensive destruction of crops (43, 47). However, the mechanisms by which B. thuringiensis ␦-endotoxins act against locusts remain unclear.…”

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Novel Bacillus thuringiensis δ-Endotoxin Active against Locusta migratoria manilensis

Lei

Dan

et al. 2011

Appl Environ Microbiol

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A novel ␦-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The ␦-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 ␣-helices (domain I); 213 residues forming three antiparallel ␤-sheets (domain II); and 134 residues forming a ␤-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC 50 s) were 8.98 g/ml for the expressed Cry7Ca1, 0.87 g/ml for the activated toxin 1, and 4.43 g/ml for the activated toxin 2. The ␦-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.Bacillus thuringiensis is a spore-forming entomopathogenic bacterium that produces parasporal crystals composed of proteins called ␦-endotoxins during sporulation (8,19,36,45). It is reported that B. thuringiensis exhibits specific activity against Lepidoptera, Diptera, Coleoptera, Homoptera, Hymenoptera, Mallophaga, nematodes, and other invertebrates (2,7,9,24,44). B. thuringiensis is a uniquely specific, safe, and effective biological insecticide that has been used for more than 50 years (3,30). The increased use of B. thuringiensis in biological control and its potential application in medicine have encouraged researchers to find novel strains with different toxic spectra or high specific activity and new functional genes of B. thuringiensis (13,22,26,28).There are two known classes of ␦-endotoxins in B. thuringiensis: the insect-specific insecticidal crystal (Cry) proteins and the Diptera-specific cytolytic (Cyt) proteins. The Cry proteins are encoded by cry genes, include more than 480 different genes belonging to 124 subgroups of 60 groups, and show variable degrees of sequence homology (see the B. thuringiensis [Bt] toxin nomenclature website at http://www.lifesci.sussex.ac .uk/home/Neil_Crickmore/Bt/). Most of the Cry proteins contain five conserved sequence blocks acco...

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“…It produces spherical parasporal crystals during the stationary phase of its growth cycle, and it is highly toxic to lepidopterous and dipterous insects (4,5).…”

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Complete Genome Sequence of Bacillus thuringiensis Serovar Sichuansis Strain MC28

Guan

Dai

et al. 2012

J Bacteriol

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cBacillus thuringiensis is an important microbial insecticide used in the control of agricultural pests. Here we report the finished, annotated genome sequence of Bacillus thuringiensis serovar Sichuansis strain MC28, which can form parasporal crystals consisting of Cry4Cc1, Cry30Fa1, Cry53Ab1, Cry54Aa1, Cry54Ab1, Cry68Aa1, Cry69Aa1, Cry69Aa2, Cry70Ba1, Cyt1Da1, and Cyt2Aa3. It is also highly toxic to lepidopterous and dipterous insects.

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Cloning and Characterization of Two Novel Crystal Protein Genes, cry54Aa1 and cry30Fa1, from Bacillus thuringiensis Strain BtMC28

Cited by 28 publications

References 17 publications

Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

Novel Bacillus thuringiensis δ-Endotoxin Active against Locusta migratoria manilensis

Complete Genome Sequence of Bacillus thuringiensis Serovar Sichuansis Strain MC28

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