Characterization of the Fumarase Gene of Bacillus subtilis 168 Cloned and Expressed in Escherichia coli K12

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Abstract
The fumarase (citG) gene of B. subtilis 168 was identified in a collection of .lambda. phages carrying EcoRI-generated fragments of B. subtilis DNA. Regions of the cloned DNA were subcloned into plasmid vectors, and the ability of prophages and multicopy plasmids to complement E. coli and B. subtilis fumarase mutations was examined. Two EcoRI fragments of 1.5 and 5.1 kilobases 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 .apprx. 1/10 of that from the normal E. coli gene; the level is increased by expression from a pBR322-derived multicopy plasmid. The citG gene was located within the cloned DNA by transposon mutagenesis and by expression studies, which also identified a polypeptide of MW 49,000 as the product of the citG gene. The properties of a truncated derivative of this polypeptide indicated the direction of transcription of the citG gene.