Molecular analysis of the mosquito larvicidal toxins of Bacillus sphaericus 1593M.

Bacillus sphaericus produces a sporulation associated toxin specific for the larvae of dipteran insects: mosquitoes that are vectors for the transmission of human diseases such as malaria and filariasis. Two dissimilar DNA sequences conferring larvicidal activity on recombinant Escherichia coli had previously been cloned from B. sphaericus 1593M (Souza at al., (1988), J. Biotechnol., 7, 71-82). Proteins encoded by one of these DNA sequences are studied in this thesis. The DNA sequence cloned from B. sphaericus 1593M is here shown to encode two proteins of 41 and 59KDa that are together required for toxicity to mosquito larvae. These proteins were previously hypothesized to exist in the crystal of B. sphaericus as high molecular weight oligomers (Baumann et al., (1985), J. Bacteriol., 163, 738-747). I now show that these two proteins are distinct from the high molecular weight protein which appears to constitute the majority species of the crystal. I have identified this protein as the Surface layer protein of B. sphaericus. I also show that the 59KDa species is a distinct and separate constituent of the crystal. As a prerequisite for raising antibodies to study regulation of synthesis of these proteins, purification following overproduction or secretion was investigated. Thus, attempts were made to purify the 41 and 59KDa proteins from E. coli cells using the C-terminal signal sequence of Haemolysin, a protein normally secreted from E. coli. Furthermore, attempts were made to express the gene encoding the 4lKDa protein in E. coli to a high level from PR and PL promoters. I show that this protein could not be overexpressed in this way. This was attributed to a lack of transcript termination on the vector due to the presence of a faulty fd transcription terminator downstream of the gene. The nucleotide sequence of the gene encoding the 59KDa protein was determined. This sequence was then used to design an approach which led to the overexpression of the gene from the T7 RNA polymerase-recognized 10 promoter on the plasmid pET3a. This in turn allowed the production of antibodies. The expression of the mRNA transcripts of the genes encoding the 41 and 59KDa proteins were also studied in B. sphaericus and B-galactosidase fusions were constructed to serve as "reporters" for the expression of the 41 and 59KDa proteins in E. coli and B. subtilis. I propose that the regulation of expression of the genes is complicated, and depends upon the use of two, temporally regulated promoters.