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• W2034253009 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 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. Mulliez E. Meier C. Trautwein A. Fontecave M. J. Biol. Chem. 1999; 274: 31291-31296Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This class can only operate under anaerobic conditions and has been found in archaea and eubacteria (8Torrents E. Aloy P. Gibert I. Rodriguez-Trelles F. J. Mol. Evol. 2002; 55: 138-152Crossref PubMed Scopus (79) Google Scholar). Although there are significant differences between the three RNR classes, sequence alignments and in particular comparisons of their three-dimensional structures highlights striking similarities, including conservation of functional cysteines required for catalysis (12Booker S. Licht S. Broderick J. Stubbe J. Biochemistry. 1994; 33: 12676-12685Crossref PubMed Scopus (91) Google Scholar, 13Jordan A. Torrents E. Jeanthon C. Eliasson R. Hellman U. Wernstedt C. Barbé J. Gibert I. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13487-13492Crossref PubMed Scopus (29) Google Scholar, 14Borovok I. Kreisberg-Zakarin R. Yanko M. Schreiber R. Myslovati M. Åslund F. Holmgren A. Cohen G. Aharonowitz Y. Microbiology. 2002; 148: 391-404Crossref PubMed Scopus (27) Google Scholar). All three RNR classes also share a sophisticated allosteric regulation mediated by different deoxy-and ribonucleotides, which guarantees an adequate and balanced supply of DNA precursors during DNA replication and repair. The essentiality of this enzyme makes it a good candidate for anticancer, antiviral, and antibacterial drug therapy (15Nocentini G. Crit. Rev. Oncol. Hematol. 1996; 22: 89-126Crossref PubMed Scopus (100) Google Scholar, 16Mayhew C.N. Phillips J.D. Cibull M.L. Elford H.L. Gallicchio V.S. Antivir. Chem. Chemother. 2002; 13: 305-314Crossref PubMed Scopus (10) Google Scholar, 17Tsimberidou A.M. Alvarado Y. Giles F.J. Expert Rev. Anticancer Ther. 2002; 2: 437-448Crossref PubMed Scopus (67) Google Scholar). In P. aeruginosa, the investigation of the expression and physiological function of the three different classes of RNR is important from a biomedical and physiological point of view, since such studies might contribute to a better understanding of the pathogenicity and the metabolic diversity of this species. In this study, we report that P. aeruginosa class II RNR (NrdJ) differs from all hitherto characterized RNRs in having a split nrdJ gene with two consecutive ORFs separated by 16 bp. Since class II RNR activity was earlier described in P. aeruginosa crude extracts (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google Scholar), our discovery of the split nrdJ gene prompted further studies of functional aspects of class II RNR in P. aeruginosa. We show that class I and II RNRs were differentially expressed in aerobically growing P. aeruginosa and that class II is not a cryptic enzyme but requires both ORFs (NrdJa and NrdJb) for full activity and that it can support bacterial growth in the absence of class I RNR activity. Bacterial Strains, Plasmids, and Growth Conditions—All strains and plasmids used in this study are listed in Table I. Cells were cultured on Luria-Bertani (LB) medium at 37 °C. For solid media, Bacto agar (Difco) was added to a final concentration of 1.5%. When necessary, isopropyl-1-thio-β-d-galactopyranoside (1 mm) was added to the media, and antibiotics were added to the following final concentrations: for Escherichia coli, 10 μg/ml tetracycline and 50 μg/ml ampicillin; for P. aeruginosa, 50 μg/ml tetracycline.Table IStrains, plasmids, and oligonucleotides used in this studyRelevant features/sequenceOrigin or referenceStrainsE. coliDH5αrecA1, endA1, hsdR, supE44, thi-1, gyrA96, relA1, Δ lacU169, deoR Φ 80dlacZM15StratageneS17-IrecA1, thi-1, pro, hsdR, <RP4: 2-Tc: Mu-: Km:Tn7, TpR, SmR, integrated into the chromosomeRef. 44Bagdasarian M. Lurz R. Ruckert B. Franklin F.C. Bagdasarian M.M. Frey J. Timmis K.N. Gene (Amst.). 1981; 16: 237-247Crossref PubMed Scopus (780) Google ScholarP. aeruginosaPAO1Type strain ATCC 6872PlasmidspGEM-T easyE. coli PCR product cloning, AprPromegapVLT31Broad-host-range expression systemRef. 20de Lorenzo V. Eltis L. Kessler B. Timmis K.N. Gene (Amst.). 1993; 123: 17-24Crossref PubMed Scopus (374) Google ScholarpETS131pVLT31 vector carrying the nrdJa fragmentThis workpETS133pVLT31 vector carrying the nrdJa + nrdJb fragmentThis workOligonucleotidesaF and R denote forward and reverse primers, respectively, and the number is the nucleotide sequence from the start codon. Gene-specific primers used in RT-PCR and real time PCR were designed using the Primer Express ABI Prism program (Applied Biosystem), using the available information from the published P. aeruginosa genome sequence (24).Class I RNR (nrdA and nrdB)F1-12805′-TGCCCTTCCTGAAAGTGGTCA-3′F2-28535′-GTGCAGCATCGATAACCCC-3′R1-39625′-GGTGAAGCCGCAGTAGAAG-3′R2-35675′-TGAGGCCGTCCTTGCTCTT-3′R3-14905′-TCGAAGACGCGCTTCATGAA-3′Class II RNR (nrdJa and nrdJb)F3-15485′-TGCTCCGGAACTGATCGA-3′F4-20445′-TCAAGGAGTCCGACCTGGAGA-3′R4-24305′-ACCATGTCGTTGATGGTCA-3′R5-17065′-CGGATAAGGTTGCGCGAATA-3′OP1-VLT-up5′-ATCTAGATATTGATTCCCGTCAG-3′OP2-J1-lw5′-AAAGCTTCTCAGTACTTGCCGTAATAG-3′OP4-J2-lw5′-AAGCTTCTCACCCACATTTCGAAGTG-3′16 S rDNAF5-12005′-ACACGTGCTACAATGGTCGGT-3′R6-1305′-TTCACGCAGTCGAGTTGCA-3′procCF6-4475′-CAGGCCGGGCAGTTGCTGTC-3′Ref. 29Savli H. Karadenizli A. Kolayli F. Gundes S. Ozbek U. Vahaboglu H. J. Med. Microbiol. 2003; 52: 403-408Crossref PubMed Scopus (261) Google ScholarR7-6155′-GGTCAGGCGCGAGGCTGCT-3′Ref. 29Savli H. Karadenizli A. Kolayli F. Gundes S. Ozbek U. Vahaboglu H. J. Med. Microbiol. 2003; 52: 403-408Crossref PubMed Scopus (261) Google Scholara F and R denote forward and reverse primers, respectively, and the number is the nucleotide sequence from the start codon. Gene-specific primers used in RT-PCR and real time PCR were designed using the Primer Express ABI Prism program (Applied Biosystem), using the available information from the published P. aeruginosa genome sequence (24Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3418) Google Scholar). Open table in a new tab DNA and Protein Techniques—Recombinant DNA manipulations and protein analyses were carried out according to published protocols (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Genomic DNA from P. aeruginosa PAO1 was isolated using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's specifications. RNA Extraction and Reverse Transcription-PCR—For total RNA preparation, P. aeruginosa PAO1 cultures were mixed directly with RNAprotect bacteria reagent (Qiagen). This reagent is designed for direct stabilization of bacterial RNA in vivo, and its protection from degradation during sample collection and storage (Qiagen). After mixing, samples were immediately frozen in liquid nitrogen and kept at –80 °C until RNA extraction, which took place less than a week after harvest. RNA was purified using an RNeasy kit (Qiagen) in duplicates and pooled together after the extraction. An aliquot of the culture (at A600 = 0.4) equivalent to 108 cells was used for one RNA preparation. DNA contamination was removed from the RNA preparation by treatment with RNase-free DNase (Qiagen), followed by the RNase-free DNase digestion from Ambion according to the manufacturers' instructions. The amount of the RNA was determined from its 260-nm absorption using a Smart Spec™ spectrophotometer (Bio-Rad). RNA quality was estimated from the 260/280 ratio, which was equal to 1.9–2.0 in all preparations, and from electrophoresis on a 1% agarose gel. For the RT step, 2 μg of P. aeruginosa total RNA was mixed with dNTPs (1 mm final concentration of each), and 10 μmol of specific primers in a 20-μl reaction. The reaction mixture was heated at 65 °C for 5 min followed by quick transfer to ice. After the addition of buffer, 40 units of RNase inhibitor RnaseOUT, 15 units of Thermoscript reverse transcriptase (Invitrogen), and 5 mm DTT final concentration, 20-μl reactions were incubated at 60 °C for 50 min, followed by a 5-min inactivation step at 85 °C in a Robocycler 96 (Stratagene). In control samples (–RT in Fig. 1B), reverse transcriptase was omitted from the reactions, which were run in parallel in the Robocycler. To digest the RNA at the end of the RT reaction, 2 units of E. coli RNase H were added, and reactions were incubated at 37 °C for 20 min. For the subsequent PCR amplification, 2 μl of the RT reaction (or negative control) was mixed with 0.2 mm each pair of primers, 0.2 mm of each dNTP, 5 μl of 10× PCR buffer and 1.5 units of High Expand Fidelity Taq polymerase (Roche Applied Science) in a total volume of 50 μl. For co-transcription studies of nrdAB, PCR mixtures were supplemented with Me2SO to 0.4% final concentration. PCR amplifications were carried out under the following conditions: 94 °C for 180 s, followed by 35 cycles of 60 s at 94 °C, 60 s at either 49 °C (nrdAB studies) or 56 °C (nrdJab studies), and 90 s at 72 °C and completed by 420 s at 72 °C. The RT-PCR end point products of both positive reactions (+RT) and negative controls (–RT) were run on 2% agarose gel, containing ethidium bromide, and visualized under UV light. Quantitative RT-PCR Studies—To quantify class I and class II RNR transcripts from exponential to stationary growth phase as well as after HU treatment, several different protocols from Applied Biosystems, Qiagen and Invitrogen were tested. In the final protocol 0.5 μg of total P. aeruginosa RNA was reverse transcribed in the Robocycler in a 25-μl volume with Taqman RT reagent kit (Applied Biosystems) at 48 °C using random hexamers according to the manufacturer's specifications. In the subsequent real time PCR step, 10 μl of a 10-fold diluted RT product corresponding to 20 ng of a total RNA was mixed with a 30-μl premix consisting of a ready to use qPCR Platinum® SYBR® Green qPCR SuperMix-UDG kit (Invitrogen), 0.8 μl of 50× concentrated ROX reference dye (Invitrogen), and 0.2 μm each of the selected specific primers. Negative controls (–RT) were run in parallel in each run. PCR amplifications were performed on an ABI Prism 7000 sequence detector (Applied Biosystems) in a 96-well optical reaction plate sealed with optical adhesive covers (Applied Biosystems). Before amplification, a uracil-DNA glycosylase decontamination step that prevents carryover of PCR products between wells was done at 50 °C for 120 s. The PCR amplification cycle consisted of 120 s of hot start at 95 °C, followed by 50 two-step amplification cycles of 20 s at 95 °C and 30 s at 62.5 °C. At the end of the thermocycling protocol, a melting curve analysis was done between 60 and 95 °C to confirm specific product amplification. Reactions were run in triplicates for each experimental time point and at two or more independent occasions. The results were analyzed in Microsoft Excel. Copy numbers of nrdA, nrdJa, 16 S rRNA, and proC transcripts were calculated, following the suggestions of Applied Biosystems (19Biosystems Applied User Bulletin 2: ABI PRISM 7700 Sequence Detection System. Applied Biosystems, Foster City, CA1997: 1-24Google Scholar), from calibration curves obtained from serially diluted linearized copies of plasmid DNA or cDNA (usually within the range of 102 to 107) containing either nrdJa, nrdA, 16 S rRNA, or proC target sequences. To compensate for possible variations in quantity or quality of starting RNA and/or differences in reverse transcriptase efficiencies, the number of copies of nrdJa and nrdA mRNA determined in the real time PCR experiment for each time point were then normalized on the basis of 16 S rRNA or proC levels in the same sample. Overexpression of the nrdJ Region Products—The broad-host-range expression vector pVLT31 (20de Lorenzo V. Eltis L. Kessler B. Timmis K.N. Gene (Amst.). 1993; 123: 17-24Crossref PubMed Scopus (374) Google Scholar) was used to overproduce the proteins encoded in the nrdJ genomic region. To construct pETS131, the nrdJa region was amplified by PCR using primers OP1-VLT-up and OP2-J1-lw and using genomic DNA from the P. aeruginosa PAO1 strain. The 2209-bp fragment was cloned into pGEM-T easy vector (Promega), and the XbaI and HindIII double-digested fragment containing the nrdJa region was cloned into the corresponding sites of pVLT31. A similar procedure instead using primers OP1-VLT-up and OP4-J2-lw for cloning the 2913-bp entire nrdJ region was used for the construction of plasmid pETS133. Plasmids were transferred by biparental conjugation from E. coli S17-I to P. aeruginosa PAO1 on membrane filters with early log phase E. coli donors and P. aeruginosa recipients grown overnight at 43 °C. Filters were incubated at 37 °C on LB agar for 6 h before plating onto selective LB medium plates. For expression analyses, P. aeruginosa carrying pVLT31, pETS131, and pETS133 were grown until midlog phase, and cells were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 5 h. Culture aliquots (5 ml) were centrifuged and dissolved in 1 ml of buffer (30 mm Tris-HCl, pH 7.5, 10 mm DTT, 10 mm MgCl2, and 1 mm phenylmethylsulfonyl fluoride). Protein crude extracts were obtained by sonication followed by centrifugation (14,000 rpm in a microcentrifuge). Enzyme Assay—Ribonucleotide reductase class II activity was measured with CTP as a substrate by the procedure described in Refs. 4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google Scholar and 21Thelander L. Sjöberg B-M. Eriksson S. Methods Enzymol. 1978; 51: 227-237Crossref PubMed Scopus (71) Google Scholar. Assay mixtures (50 μl) were incubated for 20 min at 25 °C and contained 50 mm Tris-HCl, pH 7.6, 20 mm CaCl2, 30 mm DTT, 30 μm adenosylcobalamin, 1 mm [3H]CTP (15,753 cpm/nmol) and 0.4 mm dATP. HU (50 mm) was added to the assay mixture to inhibit residual class Ia activity. One unit of enzyme activity corresponds to 1 nmol of dCTP formed per min. Specific activity is expressed as units/mg of total protein. Sequence Alignments and Phylogenetic Inference—Sequence similarity searches were performed online using BLASTP (version 2.2.10, NCBI), and global protein and DNA sequence alignments were created with the ClustalX program (version 1.8.1) (22Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35497) Google Scholar). For NrdJ proteins of the proteobacterial group, we concatenated the protein sequences of the split NrdJ proteins (NrdJa + NrdJb). Neighbor joining was used to recover a protein distance matrix using ClustalX software. Default protein weight matrix (Blosum series) was used for alignments, and positions with gaps were excluded. The resultant tree was visualized with TreeView 1.6.6 (23Page R.D. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar). The following GenBank™ accession numbers were used for the NrdJa and NrdJb sequences: P. aeruginosa PAO1, NP_254184.1 and NP_25483.1; P. aeruginosa UCBPP-PA14, ZP_00140329.1 and ZP_00140328.2; A. vinelandii, ZP_00090826.1 and ZP_00098027.2; M. flagellatus, ZP_00173560.1 and ZP_00173561.2; P. profundum, YP_130342.1 and YP_130343.1; Magnetococcus sp., ZP_00290088.1 and ZP_00290089.1. Genomic Organization of P. aeruginosa Class I and Class II Ribonucleotide Reductases—To study the genomic organizations of both class I and II RNR, we used the information from the complete P. aeruginosa PAO1 genome sequence (24Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3418) Google Scholar). Class I RNR consisted of nrdA (PA1156) and nrdB (PA1155) genes encoding proteins of 963 amino acids and 415 amino acids linked together by a 263-bp intergenic region (Fig. 1A). Surprisingly, analysis of the class II RNR DNA region revealed a split of the nrdJ gene into two ORFs, a longer 2205-bp ORF (PA5497) and a shorter 690-bp ORF (PA5496), encoding polypeptides of 734 amino acids and 229 amino acids, which we name NrdJa and NrdJb, respectively (Fig. 1A). Sequencing of several PCR products covering the entire nrdJ intergenic region confirmed the split of the nrdJ gene into two parts by the presence of a UGA stop codon (cf. Fig. 6B). To determine whether genes encoding class I or II RNR are co-transcribed, RNA from exponentially growing cells cultured in LB medium were isolated and reverse transcribed with a specific primer either to nrdB (R1-3962) or to nrdJb (R4-2430). Amplification of cDNA with primer pair F2–2853/R2–3567 for analyses of nrdA/nrdB or F4 –2044/R4 –2430 for nrdJa/nrdJb analyses (Fig. 1A) generated PCR products of the expected size (715 and 387 bp) and covered the entire intergenic regions of interest (Fig. 1B). Clearly, in both cases, nrdA/nrdB and nrdJa/nrdJb genes are co-transcribed. Sequence analyses of several RT-PCR products from the nrdJ region confirmed the presence also at the mRNA level of the stop codon and the 16-bp sequence separating the nrdJa and nrdJb genes. Class II Enzyme Activity Requires Both NrdJa and NrdJb— Next we asked whether both NrdJa and NrdJb proteins are expressed. As shown by RT-PCR, nrdJa and nrdJb genes are co-transcribed, but the upstream part of nrdJb had no apparent consensus sequence for a ribosome-binding site as judged from comparison with the 3′ sequence of the 16 S rRNA (GenBank™ accession number AB037545) from P. aeruginosa (25Shine J. Dalgarno L. Eur. J. Biochem. 1975; 57: 221-230Crossref PubMed Scopus (80) Google Scholar, 26Kozak M. Gene (Amst.). 1999; 234: 187-208Crossref PubMed Scopus (1129) Google Scholar). We therefore constructed two different expression derivatives by cloning either the nrdJa fragment or the entire nrdJa/nrdJb fragment into the Pseudomonas expression vector pVLT31. Expression of the protein encoded in the nrdJa fragment (pETS131 plasmid) shows a strong band around 82 kDa corresponding to the expected molecular weight of the NrdJa protein (Fig. 2, lane 3). The plasmid carrying the whole nrdJ region (pETS133) shows expression of two major proteins with molecular masses of about 82 and 25 kDa, corresponding to the NrdJa and NrdJb proteins (Fig. 2, lane 4). These results confirm that both proteins are expressed in growing cells of P. aeruginosa. In addition, no band corresponding to the molecular mass of a fused NrdJa + NrdJb polypeptide was seen in any of our expression experiments. Both NrdJa and NrdJb encode highly conserved RNR regions. NrdJa contains all the catalytic residues located at the active center of class II RNR including the three conserved cysteines (Cys-123, Cys-342, and Cys-353) needed for catalysis, and NrdJb carries the C-terminal cysteines that interact with the physiological reducing agents thioredoxin and glutaredoxin (12Booker S. Licht S. Broderick J. Stubbe J. Biochemistry. 1994; 33: 12676-12685Crossref PubMed Scopus (91) Google Scholar, 27Arnér E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2001) Google Scholar) needed for the enzyme turnover cycle. Due to the split of these two important functions of the class II RNR we a priori expected an inactive enzyme, but a class II RNR activity was previously demonstrated in a crude extract 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). We therefore used the plasmid constructs to test whether both proteins were needed for a functional class II RNR or whether the NrdJa protein was enzymatically active by itself when supplied with an artificial hydrogen donor like DTT. To avoid measuring intrinsic class I RNR activity, we used dATP as allosteric effector (negative effector of class I and positive effector of class II RNR) and included the class I-specific inhibitor HU in some assays. As shown in Table II, a crude extract of P. aeruginosa carrying the pVLT31 vector without inserts has almost no activity without the class II-specific cofactor AdoCbl, and it is only after the addition of AdoCbl that we observe a class II RNR activity of about 0.068 units/mg protein, which corresponds to the previously found activity in crude extracts of P. aeruginosa (4Jordan A. Torrents E. Sala I. Hellman U. Gibert I. Reichard P. J. Bacteriol. 1999; 181: 3974-3980Crossref PubMed Google Scholar). When NrdJa is expressed from pETS131, the specific activity increases somewhat to 0.169 units/mg when AdoCbl is present in the assay. A dramatic increase is obtained when both proteins (NrdJa and NrdJb) are expressed from plasmid pETS133, in which case the class II activity is 10-fold higher than the class II background activity in crude extract. In addition, the enzyme activity in the pETS133 extract is completely AdoCbl-dependent, because no activity is present without this cofactor.Table IIClass II ribonucleotide reductase activity of P. aeruginosa carrying overproduced NrdJa and NrdJb proteinsPlasmidsCloned genesNrdJ specific activityNo additions+HU+HU/AdoCblunits/mg proteinpVLT31None0.0060.0070.068pETS131nrdJa0.0060.0060.169pETS133nrdJa/nrdJb0.0070.0080.703 Open table in a new tab Expression of Class I and II RNR as a Function of Growth Phase—Next, we investigated the differential expression of class I (nrdA) and class II (nrdJa) RNR of P. aeruginosa during different growth phases by quantitative real time PCR. As shown in Fig. 3, expression of both nrd genes clearly are dependent on the culture growth conditions but with opposite expression pattern. Class Ia RNR (nrdA) reached its highest expression at the beginning of the exponential phase and decreased dramatically at the end of the exponential phase, where it was 6–7-fold lower compared with exponential phase. On the other hand, class II RNR (nrdJa) transcription followed an inverted pattern and was increased at the end of the exponential growth phase, and in stationary phase the transcri" @default.
• W2034253009 created "2016-06-24" @default.
• W2034253009 creator A5010854775 @default.
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