PROMOTER IDENTIFICATION AND EXPRESSION ANALYSIS OF SALMONELLA-TYPHIMURIUM AND ESCHERICHIA-COLI NRDEF OPERONS ENCODING ONE OF 2 CLASS-I RIBONUCLEOTIDE REDUCTASES PRESENT IN BOTH BACTERIA

Salmonella typhimurium and Escherichia coli cells have two different c lass I ribonucleotide reductases encoded by the nrdEF and nrdAB operon s, Despite the presence of one additional ribonucleotide reductase, th e 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 transcripti on have been analysed intensively, Nothing is known about the expressi on 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 spec ies, The +1 position was 691 bp and 692 bp upstream of the translation al start points of the nrdE genes of S. typhimurium and E. coil, respe ctively, Downstream of the +1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are prese nt in both bacteria, The synthesis of a polypeptide with a molecular m ass of 9 kDa, corresponding to the first of these two ORFs, was observ ed by using the T7 RNA polymerase expression system, Comparison of the amino acid predicted sequence of this ORF reveals a significant simil arity with glutaredoxin proteins, Competitive, reverse-transcription p olymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild-type cells, nrdEF tran scription is increased by hydroxyurea, which inhibits class I ribonucl eotide reductase activity, in both RecA(+) and RecA(-) cells, nrdA(ts) mutants show a higher level of nrdEF transcription than wild-type cel ls at either the permissive or the restrictive temperature, nrdEF expr ession was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA::Tn10 and hns::Tn10 mutations or by t he inhibition of DNA gyrase with the antibiotic novobiocin, In contras t to the nrdAB genes, the nrdEF operon is not essential to the cells b ecause nrdEF-defective mutants are viable under both aerobic and anaer obic conditions.