Biological properties of loop-replaced mutants of Bacillus thuringiensis mosquitocidal Cry4Aa

Howlader, Mohammad Tofazzal Hossain; Kagawa, Yusuke; Sakai, Hiroshi; Hayakawa, Tohru

doi:10.1016/j.jbiosc.2009.03.016

Cited by 8 publications

(11 citation statements)

References 19 publications

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“…Mutations in loops 1, 2, and 3 were introduced by a 1-day mutagenesis procedure as reported previously (17,18). The primers used for the mutagenesis are listed in Table 1.…”

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

“…The E. coli cells were harvested by centrifugation and disrupted by sonication. The GST-Cry4Aa mutant proteins were purified using glutathione-Sepharose 4B (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as reported previously (16,17,39). The protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA) with bovine serum albumin as a standard.…”

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

“…Mosquito larvae were reared from eggs that were supplied by the Research and Development Laboratory, Dainihon Jochugiku Co., Ltd., Osaka, Japan. The bioassay used has been described previously (17). GST and wild-type GST-Cry4Aa were used as negative and positive controls, respectively.…”

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

“…BBMV were prepared from 7-to 10-day-old C. pipiens fourthinstar larvae as described previously (17,32). BBMV containing 20 g proteins were mixed with 2 g of wild-type or loop mutant GST-Cry4Aa in phosphatebuffered saline containing protease inhibitor (Complete protease inhibitor cocktail tablets; Roche Applied Science, Mannheim, Germany) and then incubated for 1 h at room temperature.…”

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

“…Replacement of loops 1 and 3 of Cry4Aa also affected mosquitocidal activity, but it did not eliminate it (17). In this study, to further characterize the loops, we constructed Cry4Aa mutants in which individual amino acids in the loops were replaced with alanine and analyzed the mutants to determine their mosquitocidal activity against Culex pipiens larvae.…”

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confidence: 99%

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Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Howlader

Kagawa

Miyakawa

et al. 2010

Appl Environ Microbiol

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Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

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“…Mutations in loops 1, 2, and 3 were introduced by a 1-day mutagenesis procedure as reported previously (17,18). The primers used for the mutagenesis are listed in Table 1.…”

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

Section: Construction Of Cry4aa Loop Mutantsmentioning

confidence: 99%

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confidence: 99%

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