Amplification and Product Identification of the fnr gene of Escherichia coki

The position of a gene (fnr) that is essential for growth of Escherichia coli with fumarate or nitrate as electron acceptor was located within an 11.5 kb HindIII fragment of bacterial DNA by deletion analysis with fnr transducing phages (lambda fnr) and by sub-cloning restriction fragments into multicopy plasmids. The functional gene was isolated in a 1.65 kb BamHI-HindIII fragment of a hybrid plasmid pGS24. The fnr gene product was identified as a protein of Mr 31 000, by post-infection labelling and by the 'maxicell' method. Organisms containing the multicopy plasmid (pGS24) overproduced fumarate reductase to the same extent as cultures containing a comparable fumarate reductase plasmid (pNU31) during anaerobic growth; the fnr plasmid also overcame the repression of fumarate reductase synthesis that is normally observed during aerobic growth. Similar effects on nitrate reductase synthesis were also observed. The results support the view that the fnr gene product functions as a positive regulator or a specific sigma factor for expression of anaerobic energy-generating systems.