چكيده لاتين :
A new system is presented for the generation of recombinant Bacillus subtilis strains without antibiotic markers. This system is based on two plasmids constructed in Escherichia coli. The first plasmid pHM30 contains an incomplete hisl gene, the last gene in the histidine biosynthesis operon of B. subtilis and part of the genes yvcA and yvcB of unkown function flanking hisl at the 3-end. The spectinomycin resistance gene is inserted between hisl and the downstream yvcAB region. Transformation of 6. subtilis with this plasmid pHM30 led to spectinomycin resistant, histidine aux-otrophic strains. The integrated parts of pHM30 act like a docking station for the second plasmid pHM31. The plasmid pHM31 contains the same yvcAB region but a complete copy of the hisl gene and no antibiotic resistance marker. Heterologous genes to be expressed in B. subtilis were inserted into a multiple cloning site between hisl and the downstream region. Transformants of B.subtilis/pHM30 with pHM31 derivatives were selected on minimal medium without histidine. By double crossovers during homologous recombination the heterologous genes were integrated, replacing the defect copy of hisl and the spectinomycin resistance gene. The plasmids were also successfully applied in the chromosomal integration of the lipase gene of Bacillus thermocatenulatus under a B. subtilis glucose regulated promotor/antiterminator system.
