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-
After site-directed mutagenesis, the genes coding for the 42-and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the BaciUus subtilis vector pUE (a derivative of pUB18). The levels of expression of the gene products in B. subtilis DB104 and B. sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots. The results indicated that a higher amount of protein was produced in B. subtlis DB104. Electron microscopic examination of B. subilis DB104 and B. sphaericus 718 containing the 42-and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B. sphaericus 2362 were produced in the latter. Strains producing only the 42-or the 51-kDa protein were not toxic to larvae of C. pipiens. A mixture of both strains, a single strain producing both proteins, or a fusion of the 51-and the 42-kDa proteins was toxic. The amount ofB. subtlis DB104 containing the 42-and the 51-kDa proteins necessary to kill 50% of the larvae of C. pipiens was 5.6 ng (dry weight) of cells per ml. This value was significantly lower than that for B. sphaericus 2362 (14 ng [dry weight] per ml). Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein to primarily 39-and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa. Past studies involving purified proteins from B. sphaericus 2362 indicate an association of toxicity with the 39-kDa peptide. The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.
During sporulation, Bacillus sphaericus 2362 produces a parasporal crystalline protein which is toxic for the larvae of a number of mosquito species. Using the Eschertichia coli cloning vector Agtll, in which gene products of the inserts may be fused to I8-galactosidase, we isolated 29 bacteriophages which produced peptides reacting with antiserum to crystal protein. On the basis of restriction enzyme analyses of the recombinants and Ouchterlony immunodiffusion experiments with induced lysogens as a source of antigens, the recombinants were assigned to three groups, designated A, B, and C. Group A consisted of three clones which appeared to express all or part of the B. sphaericus toxin gene from their own promoters and one clone producing a ,-galactosidase-toxin fusion protein. The host cells of two induced recombinant lysogens of this group were toxic to larvae of Culex pipiens. A cell suspension containing 174 ng (dry weight) of the more toxic recombinant per ml killed 50% of the larvae. Both recombinants formed peptides with molecular sizes of 27, 43, and 63 kilodaltons (kDa). The antigenically related 27-and 43-kDa peptides were distinct from the 63-kDa peptide, which resembled crystals from sporulating cells of B. sphaericus in which antigenically distinct 43-and 63-kDa proteins are derived from a 125-kDa precursor. A 3.5-kilobase HindIII fragment from recombinants having toxic activity against larvae was subcloned into pGEM-3-blue. E. coli cells harboring this fragment were toxic to mosquito larvae and produced peptides of 27, 43, and 63 kDa. The distribution of the A gene among strains of B. sphaericus of different toxicities suggested that it is the sole or principal gene encoding the larvicidalcrystal protein. The two recombinants of group B and the 23 of group C were all ,-galactosidase fusion proteins, suggesting that in E. coli these genes were not readily expressed from their own promoters. The distribution of these two genes in different strains of B. sphaericus suggested that they do not have a role in the toxicity of this species to mosquito larvae.
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