Ribonucleotide Reductases of Salmonella Typhimurium: Transcriptional Regulation and Differential Role in Pathogenesis

Abstract

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses.

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis

“…However, the intracellular amount of these enzymes is also regulated at the transcriptional level; such control is of particular relevance in bacterial species possessing different types of RNRs, including E. coli, Salmonella, and Pseudomonas aeruginosa (7,8,35). B. subtilis possesses one class Ib RNR, encoded by the nrdEF operon (3, 4).…”

“…This protein represses the expression of genes encoding the class 1b RNR in E. coli and other bacteria (7,8,35). B. subtilis contains a putative ortholog of this protein encoded by ytcG.…”

“…Such transcription factors respond to changes in metabolic or environmental conditions and mediate the repression or induction of RNR-encoding genes (2, 3, 5-7). The transcrip-tional repressor NrdR controls the expression of genes encoding the three RNR classes in virtually all bacteria studied, although it exerts a stronger effect over genes encoding class Ib proteins (7,8).…”

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

“…Fe II –Fur also positively regulates certain genes, such as Fe–SOD, a process that is mediated posttranscriptionally by the small RNA RyhB, which is itself negatively regulated by Fe II –Fur. 174 In low Fe II conditions, when Fur is largely in the apo form, Fur’s affinity for its binding site decreases, and genes in its regulon, such as the nrdHIEF operon 66,175,176 and mntH , 65,177 are transcribed. MntH expression subsequently leads to Mn II import, ultimately facilitating loading of NrdF with Mn II .…”

“…This hypothesis is supported by studies of class Ia and Ib RNR mutants of the related pathogen S. Typhimurium during infection of macrophages. Gibert and coworkers 176 found that NrdEF, but not the class Ia NrdAB, plays a key role in early stages of infection (up to 6 h). However, a S. Typhimurium Δ nrdAB mutant is inviable at 24 h (once the pathogen’s virulence response has been induced 180 ) unless the macrophage is lacking Nramp1, the divalent metal transporter responsible for efflux of metals from the phagosome, and the cells are exposed to an iron chelator.…”