Amplification of fumarate reductase synthesis with lambdafrdA transducing phages and orientation of frdA gene expression
Thermoinducible lysis-defective derivatives of λfrdA phages (λG1F and λG40F) carrying the fumarate marate reductase gene of Escherichia coli, inserted in each of two possible orientations, were used to amplify fumarate reductase synthesis and study the aerobic repression of frdA gene expression. Anaerobic induction of lysogens containing λfrdA cItsQ−S− phages increased the fumarate reductase activities by up to 5 times the normal anaerobic level. Aerobic repression of frdA expression was overcome during aerobic induction and reductase activities up to twice the normal anaerobic level were produced. The rates and extents of amplification were dependent on the orientation of frdA in the λcItsQ−S− derivatives but not in the corresponding λcI−N phages. Assays for the frdA-linked ampC gene product, β-lactamase, indicated that the intact ampC gene is not incorporated in the λfrdA phages. Autoradiographic analysis of the polypeptides synthesized by U.V.-irradiated bacteria infected with λfrdA phages confirmed the identification of a polypeptide (Mr=72,000) as the frdA gene product (the large subunit of fumarate reductase). No precursor form was detected but another polypeptide (Mr=26.000) encoded by the 4.9 kb R.HindIII fragment was detected. The corresponding gene (g26) could be the structural gene (frdB) for the small subunit of fumarate reductase. The directions of transcription of the cloned frdA and g26 genes were deduced for the transducing phages λG1F (λfrdAr) and λG40F (λfrdA1) and were shown to be anticlockwise on the conventional Escherichia coli genetic map.