Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria

Abstract

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. nrdA(ts) 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.

Corynebacterium ammoniagenes class Ib ribonucleotide reductase: transcriptional regulation of an atypical genomic organization in the nrd cluster

“…Immunodetection of the nrdHIE fusions was unsuccessful, probably due to the low expression level reached by this promoter, as seen in the quantification of the nrdE and nrdF basal levels. This system differs from those seen in previously studied class Ib RNRs (Jordan et al, 1996a), where all genes (nrdHIEF) were transcribed in a single mRNA. These findings raised a considerable number of questions about the regulation of the genes found within this region.…”

“…Afterwards, the ratio of amplified target to competitor product was determined for the nrdE and nrdF genes. Variations in the ratios indicate the relative differences in initial mRNA levels between the samples and are calculated according to Jordan et al (1996a).…”

“…RNA was prepared and hybridized with denatured digoxigenin-labelled DNA probes against nrdE and nrdF as described by Jordan et al (1996a). This method was not sensitive enough to detect the nrd mRNA, indicating low transcriptional levels (data not shown).…”

“…Class Ib RNR was discovered in Salmonella typhimurium, without any apparent function (Jordan et al, 1996a) because class Ia was also present and it was shown to be the active class under standard laboratory growth conditions. Recent studies demonstrated a potential role for the class Ib enzyme under some special growth conditions (Masalha et al, 2001;Monje-Casa et al, 2001).…”

Ribonucleotide Reductase NrdR as a Novel Regulator for Motility and Chemotaxis during Adherent-Invasive Escherichia coli Infection

“…As a result, E101 can grow at 42°C only if complementary NrdAB activity is supplied in trans (31). The complementation assay was previously used to verify the activity of atypical RNRs (31)(32)(33).…”