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Salmonella typhimurium and Escherichia coli cells have two different class I ribonucleotide reductases encoded by the nrdEF and nrdAB operons. Despite the presence of one additional ribonucleotide reductase, the nrdAB-encoded enzyme is essential to the aerobic growth of the cell because nrdAB-defective mutants of both species are not viable in the presence of oxygen. Several factors controlling nrdAB gene transcription have been analysed intensively. Nothing is known about the expression of the nrdEF genes. To study this subject, and after cloning of E. coli nrdEF genes and sequencing of their 5′ ends, the promoter of this operon has been identified by primer extension in both bacterial species. The + 1 position was 691 bp and 692 bp upstream of the translational start points of the nrdE genes of S. typhimurium and E. coli, respectively. Downstream of the + 1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are present in both bacteria. The synthesis of a polypeptide with a molecular mass of 9 kDa, corresponding to the first of these two ORFs, was observed by using the T7 RNA polymerase expression system. Comparison of the amino acid predicted sequence of this ORF reveals a significant similarity with glutaredoxin proteins. Competitive, reverse-transcription polymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild-type cells. nrdEF transcription is increased by hydroxyurea, which inhibits class I ribonucleotide reductase activity, in both RecA+ and RecA cells. nrdAts mutants show a higher level of nrdEF transcription than wild-type cells at either the permissive or the restrictive temperature. nrdEF expression was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA ::Tn10 and hns ::Tn10 mutations or by the inhibition of DNA gyrase with the antibiotic novobiocin. In contrast to the nrdAB genes, the nrdEF operon is not essential to the cells because nrdEF-defective mutants are viable under both aerobic and anaerobic conditions.