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J. Biol. Chem., Vol. 280, Issue 17, 16571-16578, April 29, 2005

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Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

EXPRESSION AND TRANSCRIPTIONAL ANALYSIS OF THE AEROBIC ENZYMES^*

Eduard Torrents, Andrzej Poplawski¶, and Britt-Marie Sjöberg||

From the Department of Molecular Biology and Functional Genomics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden

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 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.

Received for publication, February 4, 2005 , and in revised form, February 17, 2005.

^* This work was supported by grants from the Swedish Cancer Society and the Swedish Foundation for Strategic Research (to B. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These two authors contributed equally to this work.

Supported by a postdoctoral fellowship from the Spanish Ministerio de Educación y Ciencia.

^¶ Supported by a postdoctoral fellowship from the Wenner-Grenska Samfundet. Present address: Dept. of Botany, Stockholm University, SE-10691 Stockholm, Sweden.

^|| To whom correspondence should be addressed: Dept. of Molecular Biology and Functional Genomics, Stockholm University, Svante Arrhenius väg 16–18 F3, SE-106 91 Stockholm, Sweden. E-mail: britt-marie.sjoberg{at}molbio.su.se.

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