Citrate Synthase Activity in Escherichia coli Harbouring Hybrid Plasmids Containing the gltA Gene

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Abstract
A hybrid plasmid, pDB2, was constructed by ligating a 3.24 kilobase (kb) EcoRI/HindIII fragment of the E. coli chromosome into pBR322. This was used to transform a gltA mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit MW was estimated as 47,000-49,000 by sodium dodecyl sulfate gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the 2 proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis, since the expression of enzyme activity in the strain haboring pDB2 was still subject to glucose repression.