Tiziana Caramori

In vivo generation of hybrids between two Bacillus thuringiensis insect-toxin-encoding genes

The parasporal crystal of Bacillus thuringiensis is composed of polypeptides highly toxic to a number of insect larvae. The structural genes (cryIA) encoding the Lepidoptera-specific toxin from different bacterial strains diverge primarily in a single hypervariable region, whereas the N-terminal and C-terminal parts of the proteins are highly conserved. In this report, we describe the generation of hybrid genes between two cryIA genes. Two truncated cryIA genes were cloned in a plasmid vector in such way as to have only the hypervariable region in common. The two truncated cryIA genes were separated by the tetracycline-resistance determinant (or part of it). In vivo recombination between the hypervariable regions of the cryIA genes reconstituted an entire hybrid cryIA gene. Direct sequence analysis of 17 recombinant plasmids identified eleven different crossover regions which did not alter the reading frame and allowed the production of eight different hybrid proteins. The recombination events were independent from the RecA function of Escherichia coli. Some of the hybrid gene products were more specific in their insecticidal action and one had acquired a new biological activity.

Sequence around the 159' region of the Bacillus subtilis genome : the pksX locus spans 33.6 kb

The nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined. Six ORFs were recognized, one of which extended for 13,341 nucleotides. Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism. At the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans wA gene, responsible for the synthesis of a polyketide pigment, were recognized. The observed similarities suggest that the 20 kb region and the previously reported 13.6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.

Sequence around the 159 degree region of the Bacillus subtilis genome: the pksX locus spans 33.6 kb.

The nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined. Six ORFs were recognized, one of which extended for 13,341 nucleotides. Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism. At the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans wA gene, responsible for the synthesis of a polyketide pigment, were recognized. The observed similarities suggest that the 20 kb region and the previously reported 13.6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.

Functional analysis of the outB gene of Bacillus subtilis

Three additional alleles of the outB gene of Bacillus subtilis, whose activity is required for spore outgrowth, were identified. The nucleotide sequence of three mutant genes was determined. Analyses of dominance-recessivity showed that the wild-type allele is dominant over the mutant ones. When the outB gene was placed under the control of the inducible spac-1 promoter, the presence of IPTG was necessary to obtain normal growth. The results suggested that the outB gene is required for growth of B. subtilis. Expression of outB from the sporulation promoter spoIID negatively affected subsequent spore outgrowth, without altering vegetative growth and sporulation.