Open AccessTargeted mutagenesis of loop residues in the receptor‐binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity
Author(s) -
Tuntitippawan Tipparat,
Boonserm Panadda,
Katzenmeier Gerd,
Angsuthanasombat Chanan
Publication year - 2005
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/j.femsle.2004.11.026
Subject(s) - bacillus thuringiensis , mutant , mutagenesis , site directed mutagenesis , wild type , biology , escherichia coli , toxin , microbiology and biotechnology , biochemistry , bacteria , gene , genetics
Loop residues in domain II of Bacillus thuringiensis Cry δ‐endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops β6–β7 (S 387 SPS 390 ), β8–β9 (S 410 , N 411 , T 413 , T 415 , E 417 and G 418 ) and β10–β11 (D 454 YNS 457 ) in domain II of the Cry4Ba mosquito‐larvicidal protein were changed individually to alanine by PCR‐based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130‐kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild‐type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild‐type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S 410 and E 417 in the β8–β9 loop, and Y 455 and N 456 in the β10–β11 loop are involved in larvicidal activity of the Cry4Ba toxin.