Molecular cloning and characterization of an insecticidal toxin from Pseudomonas taiwanensis.
An insecticidal toxin gene, tccC, was cloned from the recently discovered novel species Pseudomonas taiwanensis using degenerate PCR and genomic walking. The DNA sequence of the tccC gene (2,940 bp) has an open reading frame encoding 980 amino acids with a calculated molecular weight of 107.93 kDa. The amino acid sequence alignment showed the highest sequence identity (41.2%) with the insecticidal toxin from Pseudomonas entomophila. To examine the insecticidal functionality of the tccC gene product, TccC was heterologously expressed in Escherichia coli as a recombinant His6 fusion protein and purified by immobilized metal ion-affinity chromatography. The recombinant TccC was fed to Drosophila larvae at a concentration of 350 ppm, which induced about 60% mortality within 72 h. The recombinant TccC was stable at pH 7.0 and at 37 °C. When the pH was less than 5.0 or greater than 9.0, or temperature was greater than 55 °C, less than 20% Drosophila larvae mortality was observed. These results prove that Pseudomonas taiwanensis could be used as a source for developing novel biopesticides.
Institute of Biotechnology and Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan.
Je-Ruei
JR
Liu
Yung-Di
YD
Lin
Shying-Tzu
ST
Chang
Yi-Fang
YF
Zeng
San-Lan
SL
Wang
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004330:Drosophila / Q000187:drug effects / Q000502:physiology; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011549:Pseudomonas / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D007306:Insecticides