A STUDY OF THE INSECTICIDAL TOXINS OF BACILLUS SPHAERICUS

Ph.DDOCTOR OF PHILOSOPH

Association of Sindbis Virus Proteins with Vero Cell Cytoskeleton

The involvement of Vero cell cytoskeleton in Sindbis virus replication was studied. Cytoskeleton disrupting drugs, i. e. Vinblastine sulphate (Vin) and Cytochalasin B (CB) were used. When the infected cells were exposed to Vin immediately after infection for 9 hours, all virus-specified proteins were still present. The core protein was observed albeit at lower quantity compared with the untreated cells. Treatment with CB did not affect 93K and core proteins but PE2 and E1 were absent in the virus-specified protein profiles. Triton X-100-extracted cells also revealed modification of the remaining cytoskeleton as infection progressed. Generally the filaments became thickened and clumpy from 6 hours post infection (p. i.). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) anaylsis also revealed the core protein to be affiliated with the remaining cytoskeleton

Expression of insecticidal proteins

EP0454485A3Published Applicatio

Interaction of the Bacillus sphaericusmosquito larvicidal proteins

Genes for 51.4- and 41.9-kDa insecticidal proteins of Bacillus sphaericus were separately cloned and expressed in Escherichia coli. Both proteins were required for toxicity. Approximately equal numbers of cells containing the 51.4- and 41.9-kDa proteins produced the greatest toxicity; excess 41.9-kDa protein did not affect toxicity, whereas excess 51.4-kDa protein reduced activity. Larvae were killed when 41.9-kDa protein was fed up to 24 h after the 51.4-kDa protein, but not when the order of feeding was reversed. Radiolabelled toxins bound in approximately equal amounts to the gastric caecum and posterior midgut of Culex quinquefasciatus larvae. Radiolabelled 51.4-kDa protein was rapidly degraded by ca. 12–13 kDa in the larval gut, while 41.9-kDa protein was degraded by 1–2 kDa. Nonreduced toxin extracted from B. sphaericus produced a band on SDS–PAGE of ca. 68–74 kDa that contained both 51.4- and 41.9-kDa proteins based on sequence analysis, and a band of ca. 51 kDa that contained primarily 41.9-kDa protein. Escherichia coli containing 51.4-kDa protein enhanced toxicity of the latter eluted SDS-PAGE band. These proteins may associate very strongly, and trace amounts of 51.4-kDa protein in preparations of 41.9-kDa protein from B. sphaericus may be responsible for the previously reported toxicity of the latter