Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains.
Highly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefasciatus. To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia coli of the toxin proteins and their NH2- and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST). Toxin proteins were purified by affinity chromatography followed by cleavage from the GST carrier with thrombin. The LC50 values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42 kDa protein. Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.
Oei
C
C
Insecticidal Toxins Laboratory, National University of Singapore.
Hindley
J
J
Berry
C
C
D000330:Aedes / Q000033:anatomy & histology / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003465:Culex / Q000033:anatomy & histology / Q000378:metabolism*; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D005982:Glutathione Transferase