Ribonucleotide reductases of Salmonella Typhimurium : transcriptional regulation and differential role in pathogenesis

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

An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: α2 (NrdE), where nucleotide reduction occurs, and β2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)[subscript 6-]NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with Mn[superscript II] and incubated with fully reduced NrdI (NrdI[subscript hq]) and O[subscript 2]. Active RNR was rapidly produced with 0.25 ± 0.03 tyrosyl radical (Y·) per β2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is Mn[superscript III][subscript 2-]Y·, which we propose is generated by Mn[superscript II][subscript 2-]NrdF reacting with two equivalents of HO[subscript 2]−, produced by reduction of O[subscript 2] by NrdF-bound NrdI[subscript hq.] In the absence of NrdI[subscript hq], with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with Fe[superscript II] and incubated with O[subscript 2] in the presence or absence of NrdI[subscript hq] gave 0.2 and 0.7 Y·/β2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdI[subscript hq] hinders Fe[superscript III][subscript 2-]Y· cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the Mn[superscript III][subscript 2-]Y· cofactor, not the diferric-Y· one, is the active metallocofactor in vivo.National Institutes of Health (U.S.) (Grant number GM81393)National Defense Science and Engineering Graduate Fellowshi