Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

Abstract

The opportunistic human pathogen Pseudomonas aeruginosa is one of a few microorganisms that code for three different classes (I, II, and III) of the enzyme ribonucleotide reductase (RNR). Class II RNR of P. aeruginosa differs from all hitherto known class II enzymes by being encoded by two consecutive open reading frames denoted nrdJa and nrdJb and separated by 16 bp. Split nrdJ genes were also found in the few other ␥-proteobacteria that code for a class II RNR. Interestingly, the two genes encoding the split nrdJ in P. aeruginosa were co-transcribed, and both proteins were expressed. Exponentially growing aerobic cultures were predominantly expressing the class I RNR (encoded by the nrdAB operon) compared with the class II RNR (encoded by the nrdJab operon). Upon entry to stationary phase, the relative amount of nrdJa transcript increased about 6 -7-fold concomitant with a 6-fold decrease in the relative amount of nrdA transcript. Hydroxyurea treatment known to knock out the activity of class I RNR caused strict growth inhibition of P. aeruginosa unless 5-deoxyadenosylcobalamin, a cofactor specifically required for activity of class II RNRs, was added to the rich medium. Rescue of the hydroxyureatreated cells in the presence of the vitamin B 12 cofactor strongly implies that P. aeruginosa produces a functionally active NrdJ protein. Biochemical studies showed for the first time that presence of both NrdJa and NrdJb subunits were absolutely essential for enzyme activity. Based on combined genetic and biochemical results, we suggest that the two-component class II RNR in P. aeruginosa is primarily used for DNA repair and/or possibly DNA replication at low oxygen tension.

Shift in Ribonucleotide Reductase Gene Expression in Pseudomonas aeruginosa during Infection

“…The nrdA and nrdB genes are essential when P. aeruginosa is growing aerobically in LB medium (29). This result is also supported by the fact that no nrdAB mutants were found in a nonredundant transposon insertion in P. aeruginosa PA14 (19), demonstrating the essentiality of the nrdAB genes under aerobic conditions.…”

“…Hydroxyurea is a potent class I RNR inhibitor with a tyrosyl radical-scavenging activity (31), and it mimics class I RNR deficiency in P. aeruginosa (15,29). AdoCbl is a cofactor required for class II RNR activity (29). Aerobically, the wild type and the ⌬nrdJ and ⌬nrdD mutants alone and/or with AdoCbl showed the same growth rate, reaching an A 540 of Ϸ2.5 after 21 h (Table 3; see Fig.…”

“…S1 in the supplemental material) with and without hydroxyurea (HU) and 5Ј-deoxyadenosylcobalamin (AdoCbl). Hydroxyurea is a potent class I RNR inhibitor with a tyrosyl radical-scavenging activity (31), and it mimics class I RNR deficiency in P. aeruginosa (15,29). AdoCbl is a cofactor required for class II RNR activity (29).…”

“…The tetracycline resistance (Tc r ) gene was retrieved from the plasmid pUTminiTn5-Tc (8) by digestion with SmaI and inserted into each deleted nrd gene previously digested with BamHI and end filled with Klenow (Fermentas). The nrd deletion alleles with tetracycline resistance were cloned into the pEX18Gm vector previously digested with HindIII and SacI, generating plasmids pETS147, pETS148, and pETS149, and introduced into P. aeruginosa PAO1 by conjugation (29). Corresponding chromosomal deletions were identified following counterselection on tetracycline-and sucrose-containing medium (13).…”

“…Interestingly Pseudomonas aeruginosa contains functional genes for all three RNR classes (I, II, and III) transcribed in separate operons; the three classes are encoded by the nrdAB, nrdJab, and nrdDG genes, respectively (15). In previous work we have characterized the aerobic expression of the nrdA and nrdJ genes as well as their biochemical and biophysical properties (29,32). However, the roles of the different RNR classes in P. aeruginosa pathogenesis remain unknown.…”