Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences

Abstract

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.

Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae

“…Some of the taxa within B. sphaericus can be distinguished by using test methods based on carbon-source utilization rather than acid production from carbohydrates (de Barjac et al, 1980;Baumann et al, 1984) but the use of such methods alone does not enable complete phenotypic demarcation of the DNA homology groups. The combination of carbon-source utilization tests and other physiological and biochemical characters used in this study has provided a phenotypic classification that is completely compatible with that based on DNA homology and has enabled the separation of several clusters (e.g.…”

Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity

“…Furthermore, the fact that the proteins corresponding to the 42-kDa purified toxin from two different potent strains are each active by itself (Baumann et al 1985;Sgarella and Szulmajster 1987) indicates that a transient exposure of the 42-kDa protein to the 51-kDa one may be sufficient to stimulate the toxin activity.…”

“…In a recent study Broadwell and Baumann (1987) have found that in the larval midguts the 43-kDa protein previously isolated (Baumann et al 1985) is processed to a stable form missing 10 N-terminal and about 20 C-terminal residues.…”

“…Following purification, toxicity was found to be associated with the 41.9-kDa protein (Baumann et al 1985;Sgarella and Szulmajster 1987). Upon expression of the toxin genes in Escherichia coli, the recombinant-produced toxin is active only when the 51.4-kDa and 41.9-kDa proteins are either co-expressed in the same cell or produced in separate cultures and mixed prior to testing de la Torre et al 1989).…”

Exploring biodegradable polymer production from marine microbes

“…The advantage of marine bacteria as a source of biosynthesizing PHA production in marine condition is to avoid contamination from salt-water resistance microbes and marine water is directly filtered and used as industrial large scale medium for production of PHAs [66]. In 1971, 143 marine strains had isolated genus of Beneckea and 3 strains were capable of producing PHB and first ever to be reported from marine source [62]. V. marinofulvus PHB…”