Cloning, sequencing and characterization of Fnr from Klebsiella pneumoniae

Abstract

The transcription factor Fnr (fumarate nitrate reductase regulator) globally regulates gene expression in response to oxygen deprivation in Escherichia coli. We report here the cloning and sequencing of the fnr gene from the facultative anaerobic bacterium Klebsiella pneumoniae M5al, another member of the enteric bacteria. The deduced amino acid sequence of K. pneumoniae fnr showed very high similarity (98% amino acid identity) to the Fnr protein from E. coli and contained the four essential cysteine residues which are presumed to build the oxygen-sensing [4Fe4S](+2) center. Transfer of the K. pneumoniae gene to a fnr mutant of E. coli complemented the mutation and permitted synthesis of nitrate reductase and fumarate reductase during anaerobic growth. A gene fusion between K. pneumoniae fnr and glutathione S-transferase was constructed and expressed in E. coli under anaerobic conditions in order to make the protein available in preparative amounts. The overproduced protein was purified by glutathione-Sepharose 4B affinity chromatography in the absence of oxygen, and biochemically characterized.