Deletion of the Bacillus subtilis sdh operon

Plasmid pKIM2 carries the Bacillus subtilis sdh operon and adjacent regions of the bacterial chromosome. The plasmid replicates in Escherichia coli but not in B. subtilis. Different portions of the sdh operon were removed from pKIM2 and replaced by a cat gene derived from pC194. A series of plasmids carrying sdh deletions was thus derived. Plasmid DNA was linearized at restriction sites within the vector part and used to transform B. subtilis to chloramphenicol resistance. The majority of the transformants had a succinate dehydrogenase-negative phenotype and were deleted in the sdh operon as verified by Southern blotting. The B. subtilis deletion mutants were used to determine the functional integrity of cloned sdh genes.