Effects on larvicidal activity of single proline substitutions in alpha3 or alpha4 of the Bacillus thuringiensis Cry4B toxin.
The possible role of alpha-helices 3 and 4 in toxicity of the dipteran-active Bacillus thuringiensis Cry4B delta-endotoxin was investigated by employing proline substitutions via site-directed mutagenesis. Similar to the wild-type Cry4B, the mutant toxins were over-expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito-larvae. However, single proline replacements near the center of helix 3 (V119P) and at the N-terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild-type toxin was significantly reduced by two-hour preincubation with the non-toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.
Uawithya
P
P
Center for Molecular Genetics and Genetic Engineering, Institute of Science and Technology for Research and Development, Mahidol University, Nakornpathom, Thailand.
Tuntitippawan
T
T
Katzenmeier
G
G
Panyim
S
S
Angsuthanasombat
C
C
D000330:Aedes; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics*; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins; D007814:Larva; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010572:Pest Control, Biological; D011392:Proline / Q000737:chemistry* / Q000235:genetics*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D011392:Proline