Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

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 γ-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. 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 γ-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. Pseudomonas aeruginosa is a ubiquitous Gram-negative γ-proteobacterium capable of causing disease in plants, animals, and humans. It is an opportunistic pathogen and the leading source of nosocomial infections, causing disease in a wide range of immunocompromised patients. It is also the common cause of chronic lung infections in individuals suffering from cystic fibrosis (1Lyczak J.B. Cannon C.L. Pier G.B. Microbes Infect. 2000; 2: 1051-1060Crossref PubMed Scopus (995) Google Scholar). P. aeruginosa is not only noted for its pathogenicity but for its environmental versatility, because it is able to grow with very simple nutrient requirements and uses a huge number of different metabolic pathways (2de Lorenzo V. Environ. Microbiol. 2000; 2: 349-354Crossref PubMed Scopus (12) Google Scholar). Although the bacterium is respiratory and never fermentative, it will grow in the absence of oxygen if nitrate is available as a respiratory electron acceptor (3Zumft W.G. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2897) Google Scholar). Interestingly, P. aeruginosa is one of a few organisms to encode in its genome three different classes (Ia, II, and III) of the enzyme ribonucleotide reductase (RNR) 1The abbreviations used are: RNR, ribonucleotide reductase; AdoCbl, 5′-deoxyadenosylcobalamin; DTT, dithiothreitol; HU, hydroxyurea; ORF, open reading frame; RT, reverse transcription. (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google Scholar). This essential enzyme catalyzes the reduction of ribonucleotides to the corresponding 2′-deoxyribonucleotides via a radical-dependent mechanism, thereby providing cells with the necessary building blocks for DNA synthesis (5Sjöberg B-M. Struct. Bonding. 1997; 88: 139-173Crossref Google Scholar). All known RNRs can be divided into three classes (I, II, and III) based on structural differences, metallocofactor requirements, and mechanisms used for radical generation (5Sjöberg B-M. Struct. Bonding. 1997; 88: 139-173Crossref Google Scholar, 6Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (621) Google Scholar, 7Eklund H. Uhlin U. Färnegårdh M. Logan D.T. Nordlund P. Prog. Biophys. Mol. Biol. 2001; 77: 177-268Crossref PubMed Scopus (264) Google Scholar). Class I RNRs, encoded by the nrdA and nrdB genes, are found in both prokaryotic and eukaryotic organisms. This class has a tetrameric (α2β2) structure consisting of two homodimeric proteins: R1 (α2), with the active site and allosteric sites, and R2 (β2), with a stable tyrosyl radical essential for catalysis and linked to a diiron-oxo center required for radical generation. The activity of class I RNR is restricted to aerobic conditions. Class II RNRs, encoded by the nrdJ gene, consist of a single polypeptide chain and are either monomeric (α) or homodimeric (α2). Class II RNRs use adenosylcobalamin (AdoCbl) in the radical generation process and operate both under aerobic and anaerobic conditions. This class has been found in archaea, eubacteria, and some lower eukaryotes (8Torrents E. Aloy P. Gibert I. Rodriguez-Trelles F. J. Mol. Evol. 2002; 55: 138-152Crossref PubMed Scopus (79) Google Scholar). Class III RNRs, encoded by the nrdD gene, are homodimeric (α2) and carry a stable but oxygen-sensitive glycyl radical (9Sun X.Y. Ollagnier S. Schmidt P.P. Atta M. Mulliez E. Lepape L. Eliasson R. Gräslund A. Fontecave M. Reichard P. Sjöberg B-M. J. Biol. Chem. 1996; 271: 6827-6831Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). They require a specific activase, encoded by the nrdG (β2) gene, that uses S-adenosylmethionine to generate the glycyl radical (10Eliasson R. Fontecave M. Jörnvall H. Krook M. Pontis E. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3314-3318Crossref PubMed Scopus (51) Google Scholar, 11Tamarit J. 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In P. aeruginosa, the investigation of the and of the three different classes of RNR is from a and of studies to a of the pathogenicity and the metabolic of this In this we that P. aeruginosa class II RNR differs from all hitherto RNRs in a split nrdJ with two consecutive separated by 16 bp. class II RNR activity was in P. aeruginosa (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google of the split nrdJ studies of functional of class II RNR in P. that class I and II RNRs were in growing P. aeruginosa and that class II is not a enzyme but both and for activity and that it can growth in the absence of class I RNR activity. and and used in this are in I. were on at was added to a of was added to the and were added to the for and for P. aeruginosa, and used in this or into the M. R. J. PubMed Scopus Google Lorenzo V. L. PubMed Scopus Google the nrdJa the nrdJa nrdJb and and reverse and the number is the sequence from the used in and time were the the available from the P. aeruginosa genome sequence I RNR and II RNR and H. A. F. S. U. H. J. Microbiol. PubMed Scopus Google H. A. F. S. U. H. J. Microbiol. PubMed Scopus Google and and reverse and the number is the sequence from the used in and time were the the available from the P. aeruginosa genome sequence P. M. L. E. S. Y. A. R. 2000; PubMed Scopus Google Scholar). in a DNA and DNA and were out to J. Scholar). DNA from P. aeruginosa was the to the and P. aeruginosa cultures were with This is for of in and its from during and were in and at which a was an in and the of the to cells was used for one DNA was from the by treatment with by the from to the The amount of the was from its a was from the which was to in all and from on a the of P. aeruginosa was with of and of specific in a The was at for by to the of of of reverse and were at for by a at in a In in reverse was from the which were in in the the at the of the of E. were and were at for the of the was with of of of and of in a of studies of were with to were out under the for by of at at either or and at and by at The of both and were on and under class I and class II RNR from to stationary growth as as different from and were In the of P. aeruginosa was reverse in the in a with at to the In the time of a corresponding to of a was with a consisting of a to use of and of the specific were in in were on an sequence in a with a that of between was at for The of of at by of at and at the of the a was between and to specific were in for time and at two or The were in of 16 and were the of 2: from from of DNA or the range of to either 16 or for in or of and/or differences in reverse the number of of nrdJa and nrdA in the time for time were on the of 16 or in the of the nrdJ Lorenzo V. L. PubMed Scopus Google was used to the proteins encoded in the nrdJ the nrdJa was by and and DNA from the P. aeruginosa The was into and the and the nrdJa was into the corresponding of and for the nrdJ was used for the of were by from E. to P. aeruginosa on with E. and P. aeruginosa at were at on for P. aeruginosa and were phase, and cells were with for were and in of DTT, and were by by in a reductase class II activity was with as a by the in A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google and L. Sjöberg B-M. S. PubMed Scopus Google were for at and DTT, and was added to the to class activity. of enzyme activity to of activity is as of protein. and were and and DNA sequence alignments were with the J.D. F. F. 1997; PubMed Scopus Google Scholar). NrdJ proteins of the we the of the split NrdJ proteins was used to a was used for and with were The was with 1996; Google Scholar). The were used for the NrdJa and NrdJb P. aeruginosa and P. aeruginosa and A. and M. and P. and and of P. aeruginosa Class I and Class II the of both class I and II RNR, we used the from the P. aeruginosa genome sequence P. M. L. E. S. Y. A. R. 2000; PubMed Scopus Google Scholar). Class I RNR of nrdA and nrdB genes encoding proteins of and linked by a of the class II RNR DNA a split of the nrdJ into two a and a encoding of and which we NrdJa and of the nrdJ the split of the nrdJ into two by the presence of a genes encoding class I or II RNR are co-transcribed, from growing cells in were and reverse with a specific either to nrdB or to nrdJb of with for of or for of the and and the of in both and genes are of from the nrdJ the presence also at the of the and the sequence the nrdJa and nrdJb Class II NrdJa and we both NrdJa and NrdJb proteins are expressed. by nrdJa and nrdJb genes are co-transcribed, but the of nrdJb sequence for a site as from with the sequence of the 16 number from P. aeruginosa J. L. J. Biochem. PubMed Scopus Google Scholar, M. 1999; PubMed Scopus Google Scholar). two different by either the nrdJa or the into the Pseudomonas of the encoded in the nrdJa a corresponding to the of the NrdJa The the nrdJ of two proteins with of about and corresponding to the NrdJa and NrdJb proteins that both proteins are in growing cells of P. In corresponding to the of a NrdJa NrdJb polypeptide was in of NrdJa and NrdJb encode RNR NrdJa all the at the active center of class II RNR including the three cysteines and for and NrdJb the cysteines that with the and (12Booker S. Licht S. Broderick J. Stubbe J. Biochemistry. 1994; 33: 12676-12685Crossref PubMed Scopus (91) Google Scholar, Holmgren A. J. Biochem. 2000; PubMed Scopus Google for the enzyme to the split of two of the class II RNR we a an but a class II RNR activity was in a of P. aeruginosa cells (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google Scholar). used the to both proteins were for a functional class II RNR or the NrdJa was active by with an class I RNR we used as allosteric of class I and of class II and the class in some in II, a of P. aeruginosa the has activity the class cofactor AdoCbl, and it is only the of that we a class II RNR activity of about which to the found activity in of P. aeruginosa (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google Scholar). NrdJa is from the specific activity to is in the is both proteins and are from in which the class II activity is the class II activity in In the enzyme activity in the is because activity is this II ribonucleotide reductase activity of P. aeruginosa NrdJa and NrdJb specific in a of Class I and II RNR as a of we the of class I and class II RNR of P. aeruginosa during different growth by time in of both genes are on the growth conditions but with Class RNR its at the of the and at the of the phase, it was 6–7-fold lower compared with the other class II RNR an and was increased at the of the growth phase, and in stationary the was 6-fold in the of differs between the two the of nrdA a of about 16 the of the nrdJ was about 16 for of P. aeruginosa in the of both class I and II genes are and both enzymes are active under growth conditions (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google we not if both classes are for of P. the class II RNR growth in the absence of class I activity. we used the radical to knock out class I activity and added the cofactor to class II activity. 16 of on at a first of the that not on P. aeruginosa compared with the with HU, showed growth to the inhibition of class I RNR, that class I RNR growth of P. aeruginosa on Interestingly, both and showed growth for both and of the was for an growth were The of on P. aeruginosa was and was compared with in the presence of a striking of and showed growth in the and some growth in the The not as a cofactor for class II of not that P. aeruginosa can and to the required of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Hydroxyurea of Class I and II RNR under aeruginosa was in and in of the was with HU, of the was with AdoCbl, and was was from at different and the of nrdA and nrdJ were by time to in the 16 used for in we used the for in this of both and cultures of the time of of was to be stable under a of growth conditions and was to be in studies in Pseudomonas H. A. F. S. U. H. J. Microbiol. PubMed Scopus Google Scholar). the of of nrdA and nrdJa the of and to the The of both genes was increased the of and with of 6–7-fold for nrdA and for nrdJ Interestingly, of class II RNR to and the the of class I RNR to the treatment of P. aeruginosa in of class II RNR compared with the class I RNR at all time P. aeruginosa, and a few are in that genes for three different classes of RNR (Ia, II, and III) in their P. aeruginosa is also from other class in a class II that is split into two consecutive genes and In the we on the two P. aeruginosa RNR classes that under aerobic conditions and and the active of the class II enzyme and the of class I and II RNR during aerobic growth of P. first the class II RNR in P. aeruginosa a and known of the class II RNR are split in the P. aeruginosa genome by a and the two open reading frames and are separated by a an class II RNR enzyme activity in of P. aeruginosa (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. 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Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google sequence for an allosteric activity site was in the of It was that a class II RNR at the is active in the presence of R. Pontis E. A. Reichard P. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). we that the P. aeruginosa NrdJa enzyme activity with in the presence of which also all the The P. aeruginosa class II RNR only enzyme activity both NrdJa and NrdJb proteins are This is the first of a class II RNR that a two-component for enzyme activity. Class RNRs require both the active and the for a functional Class III RNRs require the to generate the glycyl radical on the has the glycyl this can E. Eliasson R. H. Gräslund A. Reichard P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google the active of class III RNR only of Class II RNRs require the vitamin B12 for activity. The three-dimensional structures for the class II enzymes from and that the cofactor to the active site G. 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Evol. 2002; 55: 138-152Crossref PubMed Scopus (79) Google and the only class II RNR found in this is the split The suggest that the split of the nrdJ has because all split genes are The sequence are the split compared with other at the DNA of the split nrdJ that also the of the is all of The on the other in 16 to and in sequence between the nrdJb genes is not about of their not The that the split class II RNR is active is a that it is essential to P. that is under growth conditions the split nrdJ is and it can growth of P. that both the nrdAB and the nrdJab are in growing P. time of the for class and showed that their are growth as is for of genes Mol. Microbiol. 1998; PubMed Scopus Google Scholar, E. I. Gibert I. Microbiology. 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Cohen G. Mol. Microbiol. PubMed Scopus Google Scholar). This is for the of class I and II RNRs in S. are found the the is ubiquitous and can be found for the in other microorganisms I. M. Schreiber R. Aharonowitz Y. Cohen G. Mol. Microbiol. PubMed Scopus Google Scholar). P. aeruginosa an to the genes for I. M. Schreiber R. Aharonowitz Y. Cohen G. Mol. Microbiol. PubMed Scopus Google Scholar, S. J. Biochem. 2002; PubMed Scopus Google Scholar). It is to on the of class I and II RNR is in P. aeruginosa, and is necessary to is in the of the RNRs also in this It is known that cultures of P. aeruginosa a of a oxygen conditions Microbiology. 2002; 148: PubMed Scopus Google Scholar). class II RNR can under conditions. Based on combined genetic and biochemical results, we that class I RNR aerobic DNA replication and that the two-component class II RNR is used for repair and/or possibly replication at low oxygen tension. are the of the split nrdJ in P. aeruginosa during of eukaryotic organisms. are to Lorenzo for the of some and used in this and to for