Characterization of the fumarase gene of Bacillus subtilis 168 cloned and expressed in Escherichia coli K12.

The fumarase (citG) gene of Bacillus subtilis 168 has been identified in a collection of lambda phages carrying EcoRI-generated fragments of B. subtilis DNA. Regions of the cloned DNA have been subcloned into plasmid vectors, and the ability of prophages and multicopy plasmids to complement Escherichia coli and B. subtilis fumarate mutations has been examined. Two EcoRI fragments of 1.5 and 5.1 kb are both required for fumarase expression in E. coli and B. subtilis. The level of fumarase activity from a single copy of the B. subtilis citG gene expressed in E. coli is approximately one-tenth of that from the normal E. coli gene; the level is increased by expression from a pBR322-derived multicopy plasmid. The citG gene has been located within the cloned DNA by transposon mutagenesis and by expression studies, which have also identified a polypeptide of Mr 49000 as the product of the citG gene. The properties of a truncated derivative of this polypeptide have indicated the direction of transcription of the citG gene.