Two proteins mediate class II ribonucleotide reductase activity in Pseudomonas aeruginosa: expression and transcriptional analysis of the aerobic enzymes.

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 gamma-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 hydroxyurea-treated cells in the presence of the vitamin B12 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.