Sequence analysis of the mosquitocidal toxin genes encoding 51.4- and 41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297

Abstract

The nucleotide sequences of a 3,479-b.Ise-pair HindIII DNA fragment from Bacillus sphaericus 2362 and a 2,940-base-pair fragment from strain 2297 were determined; only minor differences were detected between them. Each contained two open reading frames coding for proteins of 51.4 and 41.9 kilodaltons. Both proteins were required for toxicity to larvae of the mosquito Culex pipiens.The parasporal crystal synthesized by sporulating cultures of Bacillus sphaericus is toxic to larvae of a number of disease-transmitting mosquito species (27). The predominant proteins in crystals obtained from 48-h cultures of B. sphaericus 2297 and 2362 have molecular masses of 125, 110, 63, and 43 kilodaltons (kDa) as estimated from their migrations in sodium dodecyl sulfate-polyacrylamide gels (3, 5). The 110-, 63-, and 43-kDa proteins from strain 2362 have been purified, and only the 110-and 43-kDa proteins are toxic to larvae of Culex pipiens (3, 5). Immunological analyses with antisera prepared against the 63-and 43-kDa proteins indicated that these molecules are antigenically distinct, since they have no detectable cross-reactivity (3). From the observation that both antisera reacted with the 110-and 125-kDa proteins, we inferred that the 125-kDa protein was the precursor of the 110-kDa peptide which, in turn, was degraded to the 63-and 43-kDa peptides. Our data on the kinetics of synthesis of crystal proteins during sporulation (5), as well as the cloning and expression in Escherichia coli of the genes coding for the crystal proteins (2), were interpreted in line with these initial observations. Sequencing of a 3.5-kilobase DNA fragment coding for the 63-and 43-kDa proteins of B. sphaericus 2362 showed that this interpretation is untenable, since the two are on separate genes. Recently, the DNAs coding for the 43-kDa proteins from B. sphaericus 1593 and 2362 have been cloned into E. coli (4, 12) by using oligonucleotide probes based on the N-terminal sequence data for the 43-kDa protein of strain 2362 (3) to detect the appropriate recombinants. From their DNA sequences it was concluded that the 43-kDa peptide does not arise from a higher-molecular-weight precursor (4, 12).In the present communication, we present the sequences of the DNAs coding for the 63-and 43-kDa proteins from B. sphaericus 2297 and 2362. Since the deduced molecular masses of these two proteins are 51.4 and 41.9 kDa, respec-

Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains

“…Plasmids pRK42, pRK37 and pCC2297-F, encoding the deletion derivatives of the 51 kDa protein, and plasmids pME31 and pMN174, encoding the 42 kDa protein and its deletion derivative, have been described elsewhere . Plasmid pRK24, containing a DNA fragment [nucleotides 509-1 979 according to the published sequence of Baumann et al (1988)], encoding the 51 kDa protein (minus the first four codons), was generated as part of the pRK series . Plasmid pME23 contains a gene fragment (nucleotides 2023-3257) which encodes the 42 kDa protein (minus the first four codons) and was generated as part of the pME series ).…”

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush‐border membranes from mosquito larvae

“…These crystals contain two major polypeptides of 41.9 kDa and 51.4 kDa, as deduced from DNA sequence data (Hindley and Berry, 1987;Baumann et al, 1988;Arapinis et al, 1988; for complete review see Baumann et al, 1991). Bacillus subtilis or Escherichia coli expressing either of the genes encoding these two proteins are poorly toxic or even non-toxic for mosquito larvae (Baumann et al, 1988;de la Torre et al, 1989;Broadwell et al, 1990). However, transformants expressing both genes are fully toxic, indicating that the protein crystal of B. sphaericus acts as a binary toxin (Broadwell et al, 1990;Baumann and Baumann, 1991).…”

“…The insecticidal activity is directly related to the synthesis of parasporal protein crystals during sporulation (Davidson, 1983;Payne and Davidson, 1984;Kalfon et al, 1984;Baumann et al, 1985). These crystals contain two major polypeptides of 41.9 kDa and 51.4 kDa, as deduced from DNA sequence data (Hindley and Berry, 1987;Baumann et al, 1988;Arapinis et al, 1988; for complete review see Baumann et al, 1991). Bacillus subtilis or Escherichia coli expressing either of the genes encoding these two proteins are poorly toxic or even non-toxic for mosquito larvae (Baumann et al, 1988;de la Torre et al, 1989;Broadwell et al, 1990).…”

Crystallization and preliminary X-ray diffraction studies of the 51 kDa protein of the mosquito-larvicidal binary toxin fromBacillus sphaericus

“…The amino-acid sequence of the ®rst seven Nterminal residues of the crystallized 51 kDa protein was determined to be SLNYNLP by using automated Edman degradation on a Porton 2090E protein sequencer. This sequence corresponds to residues Ser29±Pro35 of the published toxin sequences (Baumann et al, 1988). Based upon the N-terminus sequence and the measured molecular mass, the Cterminus was deduced to be at residue His421; thus, the crystallized protein is composed of a total of 393 residues.…”

“…These two proteins are homologous, with 25% identity and four conserved regions between their sequences (Baumann et al, 1987(Baumann et al, , 1988. Although the detailed mechanism is not well understood, it has been established that the 51 kDa protein has a high af®nity for speci®c regions of the gut epithelial cell and directs the 42 kDa protein to these sites as a tightly bound complex (reviewed in Porter et al, 1993).…”