INTRODUCTION

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Ribonucleotide reductases catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides and thereby provide the building blocks for DNA synthesis. Three separate classes of reductases are known (20, 31, 35). Class I enzymes are aerobic enzymes, present in both bacteria and eukaryotes, with the enzyme from Escherichia coli as the prototype. They consist of two homodimers (R1 and R2) encoded by the nrdAB genes. R1 contains the catalytic site and also binds allosteric effectors. R2 contains stable tyrosyl radicals, essential for catalysis, and diferric iron centers. Radical generation requires molecular oxygen, and class I reductases therefore function only during aerobiosis. The two known subgroups of class I (class Ia and class Ib) both conform to the mentioned criteria; they differ in amino acid sequence and also slightly in their allosteric regulation. Bacteria with class Ib enzymes, encoded by the nrdEF genes, also contain a separate hydrogen donor, encoded by nrdH. Class Ib has so far been found exclusively in bacteria.

Class II reductases are widely spread among bacteria, both eubacteria and archaebacteria. They consist of a single polypeptide chain (nrdJ) whose function corresponds to that of protein R1. Adenosylcobalamin supplies the function of protein R2 and acts as radical generator in an oxygen-independent process.

Class III reductases (nrdDG) function only during anaerobiosis. The large homodimer (nrdD) contains, similar to protein R1, catalytic and allosteric sites but in addition also a glycyl radical, stable only in the absence of oxygen. The small homodimer (nrdG) carries an iron-sulfur cluster which together with S-adenosylmethionine and a reducing system generates the glycyl radical.

Why are there so many different reductases? No doubt, their already mentioned response to molecular oxygen plays a role. Conceivably, aerobic organisms could have either a class I or class II enzyme, anaerobic organisms could have a class II or class III enzyme, and facultative anaerobic organisms could have either a class II enzyme or both class I and class III enzymes. Whereas these are indeed the basic rules, many bacteria have genes for additional, apparently unnecessary enzymes. The first such example came from the facultative anaerobic enterobacteria. They contain, in addition to a class Ia and a class III enzyme, also chromosomal genes (nrdEF) for a fully active class Ib enzyme. Under usual laboratory conditions, these do not complement mutations in nrdAB, and their physiological function is not understood (21, 23, 26). Many more examples of apparently redundant reductases in a single organism have been brought to light by the recent explosion in bacterial genomics (19a). Almost any combination of reductase genes can occur. Thus, nrdEF (class Ib) and nrdJ (class II) are present simultaneously in Deinococcus radiodurans (24) and Mycobacterium tuberculosis (7), and nrdJ and nrdDG are found in Methanobacterium thermoautotrophicum (36), Pyrococcus furiosus, Pyrococcus horikoshii, and Porphyromonas gingivalis. Bacillus subtilis (28) and Streptococcus pyogenes each have several different nrdEF genes. Finally, the genomes of Clostridium acetobutylicum and Pseudomonas aeruginosa contain nrdAB, nrdJ, and nrdDG, i.e., genes encoding each class.

Such a diversity of genes is a challenge for our understanding the evolution of ribonucleotide reduction and needs exploration. For this purpose, the pseudomonads appear to be an appropriate subject. These bacteria were originally divided into five groups (I to V) and were all considered to be Pseudomonas species; more recently, only those belonging to group I have been classified as Pseudomonas species (30). Here, we also investigated some members of other groups, which we refer to as Pseudomonas-related species. Pseudomonas species are part of the gamma subdivision of proteobacteria, to which also enterobacteria belong. The Pseudomonas-related species fall into three subdivisions of proteobacteria. All species grow generally in air and are nonfermenters. They can also grow anaerobically in the presence of nitrate or arginine.

The presence of genes encoding members of each of the three classes of ribonucleotide reductases in P. aeruginosa raises two major questions. (i) Is this phenomenon limited to P. aeruginosa, or does it also occur in other Pseudomonas species? (ii) Are several of the genes expressed simultaneously to supply the deoxynucleotides required for DNA replication?

There is only scanty evidence on both points. The growth of P. aeruginosa belonging to the gamma subdivision was reported to be inhibited by hydroxyurea, a rather specific inhibitor of class I enzymes (15). On the other hand, another member of the gamma subdivision, Pseudomonas stutzeri, was shown to contain a B₁₂-dependent class II reductase (17).

Here, we show that the occurrence of multiple genes coding for ribonucleotide reductases is widespread among members of the Pseudomonas species. We demonstrate the presence of active class I and II enzymes in both P. aeruginosa and in P. stutzeri and describe some of their properties.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. The Pseudomonas and related strains used in this work were obtained from the Colección Española de Cultivos Tipo (Spanish National Collection of Type Cultures). They are listed in Table 1 and are classified according to the three subdivisions of proteobacteria. Bacteria were grown aerobically in Luria-Bertani (LB) liquid broth at 30°C on a bacterial shaker. For microaerophilic growth, the bacteria were grown at 30°C in a nitrate reduction broth under continuous flushing with N₂:CO₂ (96:4). One liter of broth contained 5 g of tryptone, 5 g of NaCl, 2 g of yeast extract, and 1 g of KNO₃. For DNA cloning, E. coli DH5F' (Clontech) was used and grown at 37°C in LB with ampicillin (50 mg/ml) when required.

DNA probes and Southern hybridization. For PCR amplification of fragments of P. aeruginosa nrdA, nrdJ, and nrdD, the required sequence information was found in the database (30a). The fragments were used as DNA probes for Southern hybridizations. For the nrdA fragment we used primers PAO-A-up (5'-ATGCTCGATAACGTCATCGA-3') and PAO-A-low (5'-CGACTGGGCCTGGTCGAT-3'), corresponding to peptides MLDNVID and IDQAQS, respectively; for the nrdJ fragment we used PAO-J-up (5'-ACCAACCCCTGCGGCGA-3') and PAO-J-low (5'-GATGGTCCCGGTCGGCGCGAT-3'), corresponding to peptides TNPCGE and IAPTGTI, respectively; for the nrdD fragment we used PAO-D-up (5'-CATATCCACGACCTCGA-3') and PAO-D-low (5'-GAGGAGTTGGTGTAGTA-3'), corresponding to peptides HIHDLD and YYTNSS, respectively.

Extraction and partial purification of ribonucleotide reductases. Bacteria grown to the end of the logarithmic phase were cooled on ice, and all further manipulations were carried out close to 4°C. Crude extracts were prepared as described previously (23) in a buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 10 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride, and 1.0 mg of egg white lysozyme per ml and precipitated with ammonium sulfate to 70% saturation. The dissolved precipitate was dialyzed (23) and either used directly for assay of enzyme activity or further purified by chromatography on dATP-Sepharose as described below. Extracts from bacteria grown under nitrogen were manipulated and extracted inside an anaerobic box. The ammonium sulfate step was omitted, but the extracts were dialyzed overnight inside the box before use.

Ribonucleotide reductase assay. The reduction of CDP or CTP was used as an assay (39). Incubation was at room temperature (ca. 26°C) for 20 min in a final volume of 0.05 ml containing 50 mM Tris-HCl (pH 8.0), 0.5 mM [³H]CDP or [³H]CTP (17 cpm/pmol), 10 mM MgCl₂, 20 mM DTT, and 2 mM ATP or 0.3 mM dATP (as allosteric effectors). To measure class II activity, the extract was preincubated with 5 mM hydroxyurea for 30 min, the assay mixture contained 15 µM adenosylcobalamin, and the assay was done anaerobically under dimmed light.

Other methods. Protein concentrations were determined by the Bradford assay (5), with crystalline bovine serum albumin as a standard. The NH₂-terminal sequence of the R1 protein from P. aeruginosa was determined in a PE-Applied Biosystems model 494 amino acid sequencer as instructed by the manufacturer. Analytical protein gel electrophoresis was done in denaturing polyacrylamide gels either with the Phastgel system (Amersham-Pharmacia Biotech) or with Mini Protean II (Bio-Rad). DNA manipulations were done by standard procedures (34).

	RESULTS

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Presence of genes for different ribonucleotide reductase classes in Pseudomonas species. The genome sequence of P. aeruginosa contains the genes nrdAB, nrdJ, and nrdDG for the three classes of ribonucleotide reductases (30a). Using the sequence information, we purified and digoxigenin-labeled fragments of each gene cluster by PCR as described in Materials and Methods to provide probes for Southern hybridizations used to identify homologous genes in other Pseudomonas or Pseudomonas-related species. The genomic DNA from each species was for this purpose digested with EcoRI and doubly digested with EcoRI and BamHI.

Ribonucleotide reductase activities in different species. To determine the extent to which the genes for the different classes were expressed during growth, extracts from aerobically growing bacteria were prepared from all species and analyzed for class I and II activity. P. aeruginosa, P. stutzeri, and R. pickettii were also grown microaerobically under nitrogen, and their extracts were analyzed for class I, II, and III activity. Under strict anaerobic conditions, obtained by addition of 3.2 mM sodium sulfide to the media in closed bottles, growth was insufficient.

P. aeruginosa and P. stutzeri express class I and class II enzymes simultaneously. In earlier work, P. aeruginosa was shown to contain class I activity (15) and P. stutzeri was shown to contain class II activity (17). After aerobic growth we found, instead, that extracts from each species contained both class I and class II activity (Table 2). Also, after microaerophilic growth under nitrogen, both types of activity were found (Table 2). Surprisingly, class I activity was greatly increased under these conditions. Attempts to find class III activity were unsuccessful under conditions that routinely detected class III activity in E. coli extracts.

Oxygen is continuously required for the class I enzymes. During microaerobic growth class I enzymes were expressed to much higher levels than during aerobic growth (Table 2). As demonstrated by the results of Table 3, oxygen was continuously required during the assay of class I enzymes to generate their tyrosyl radical. In this experiment, we preincubated anaerobic extracts from microaerobically grown bacteria first for 30 min, either aerobically or anaerobically, and subsequently carried out the assay, again either aerobically or anaerobically. In repeated experiments, we found that class I activity required aerobiosis during the second step, whereas class II activity was independent of oxygen.

Requirements for enzyme activity. Class I activity from P. aeruginosa and P. stutzeri was studied in extracts from microaerobically grown bacteria, as they showed the highest activity. Class II activity from P. stutzeri was studied in extracts from aerobically grown cells, as they contained the highest ratio of class II to class I activities. Class II activity depended linearly on the concentration of protein, whereas class I activity displayed S-shaped dependence (data not shown). The latter result can be attributed to the fact that the class I enzymes consist of two loosely bound proteins (NrdA and NrdB), similar to the E. coli class Ia reductase (6).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Effect of ATP (A) or dATP (B) on CDP reduction by Pseudomonas class I reductase. Extracts from microaerobically grown P. aeruginosa (0.14 mg of protein; ) or P. stutzeri (0.20 mg; ) were assayed under class I conditions except for the concentrations of ATP or dATP indicated on the abscissa.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Effect of ATP () or dATP () on CTP reduction by P. stutzeri class II reductase. An extract (0.17 mg of protein) from aerobically grown P. stutzeri was assayed under class II conditions except for the concentrations of ATP and dATP indicated on the abscissa.

Separation and purification of class I and II reductases. Affinity chromatography on dATP-Sepharose has enabled the rapid purification of many ribonucleotide reductases (3, 10, 11, 23). As both the NrdA (class I) and NrdJ (class II) proteins of the Pseudomonas reductases contain allosteric dATP-binding sites, we used this method for their purification and separation as described in Materials and Methods. When a bacterial extract at low salt concentration was chromatographed on a dATP-Sepharose column, NrdB (class I) was recovered in the flowthrough fraction, whereas NrdA (class I) and NrdJ (class II) were retained. NrdJ could be eluted with 0.5 mM ATP; NrdA required 5 mM ATP. With this procedure, NrdA from both P. aeruginosa (Fig. 3, lane 1) and P. stutzeri (lane 3) were obtained in a close to homogeneous form with subunit molecular masses of approximately 100 kDa. NrdJ from P. stutzeri was purified 31-fold but on gels showed several bands in addition to one band at close to 100 kDa that may correspond to the reductase (lane 4).

View larger version (88K): [in this window] [in a new window]   FIG. 3.   Separation by 12% polyacrylamide denaturing gel electrophoresis of the enzymes purified by dATP-Sepharose chromatography. Lane 1, P. aeruginosa NrdA; lane 2, molecular weight markers; lane 3, P. stutzeri NrdA; lane 3, P. stutzeri NrdJ. Sizes are indicated in kilodaltons.

	DISCUSSION

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Our data demonstrate that several Pseudomonas species and Pseudomonas-related species contain the genes for all three classes of ribonucleotide reductases and that two separate reductases are simultaneously active in at least two of these species. To date, when an organism was found to contain two separate active ribonucleotide reductases, the proteins were expressed under different circumstances, e.g., during aerobiosis or anaerobiosis (14, 32). The present finding of two active enzymes that are expressed in parallel poses new questions concerning the physiology, gene regulation, and evolution of ribonucleotide reduction.

How reliable are our results? Positive DNA hybridization data are probably good indicators for the existence of a gene, whereas negative data may simply indicate that the DNA sequence is too divergent to give a positive signal. It is, however, safe to conclude that many species of Pseudomonas contain the genetic information for several different ribonucleotide reductases. With respect to the expression of enzyme activity, negative results may be due to technical problems, as ribonucleotide reduction is notoriously difficult to assay in extracts from slowly growing cells; therefore, our data may underestimate the generality of simultaneous enzyme expression.

As described in the introduction, class I enzymes are divided into two subclasses, Ia and Ib, coded by nrdAB and nrdEF genes, respectively. The class I reductases of Pseudomonas belong to class Ia. For P. aeruginosa, this is apparent from the nucleotide sequences of the two genes as well as from the absence of nrdHI genes in the genome (21, 25). An additional distinctive criterion for class Ia enzymes is their inhibition by dATP, which depends on an allosteric dATP-binding site (activity site) present at the NH₂ terminus of the NrdA protein but absent from NrdE of class Ib enzymes (9, 12). The P. stutzeri enzyme is inhibited by dATP and thus conforms to this pattern. The P. aeruginosa reductase was not inhibited by dATP. Its amino acid sequence at the NH₂ terminus does, however, present many of the structural features typical for a dATP-binding site (12). Lack of inhibition by dATP despite the presence of a binding site was found previously for some other class Ia enzymes (2, 19) and also some class II enzymes (10). This observation suggests the presence of a degenerate site that during evolution has lost its function (10).

A comparison of the complete amino acid sequence of the P. aeruginosa NrdAB enzyme with that of other reductases gives surprising results. Both NrdA and NrdB show higher homologies with enzymes from eukaryotes (42% identity with mouse NrdA; 28% for NrdB) than with enzymes from closely related bacteria (27% identity with E. coli NrdA; 24% for NrdB). An exception occurs for the enzyme of an intracellular pathogenic bacterium, Chlamydia trachomatis (37), to which P. aeruginosa is 52% identical for NrdA and 57% for NrdB. Possibly related to this is our finding that the Pseudomonas enzyme required the continuous regeneration of its tyrosyl radical by oxygen during catalysis, similar to the mouse enzyme. For the E. coli NrdAB (16) and the Lactococcus lactis NrdEF (22) class I reductases, it was shown that once this radical was generated, molecular oxygen was no longer required.

The two class II reductases are allosterically regulated ribonucleoside triphosphate reductases, similar to some other microbial class II enzymes. The sequence of the P. aeruginosa enzyme contains the catalytically active cysteines, first found in the Lactobacillus leishmanii reductase (4), as well as residues for the allosteric substrate specificity site identified from the crystallographic investigation of enzyme-effector complexes of the E. coli class I reductase (12) and present also in other class II reductases (24). The NH₂ terminus does not contain the amino acids required for a dATP-binding site (12, 24), and consequently the enzymes were not inhibited by dATP.

The deduced amino acid sequence of the P. aeruginosa class III reductase includes appropriately positioned residues for the characteristic glycyl radical (38), the putative catalytically active cysteines (29, 35), and the effector-binding site (24). The DNA thus has the hallmarks of a gene coding for an active enzyme. However, we could not demonstrate a class III activity in bacterial extracts after microaerophilic growth, conditions that induce this activity in E. coli. We cannot exclude the possibility that this was the result of problems with the enzyme assay. However, microaerophilic growth induced a large increase in class I activity, similar to what was observed in an E. coli nrdD mutant strain, i.e., in a strain that lacks class III activity (16). Moreover, the finding that the assayed Pseudomonas strains did not grow under complete anaerobiosis is consistent with the lack of a catalytically active class III enzyme.

How does Pseudomonas employ its class I and II reductases? Why is it that the oxygen-requiring class I reductase is primarily increased during microaerobiosis and not the class II enzyme that works equally well in the absence of oxygen? The two enzymes use different substrates. The class I enzyme is a diphosphate reductase, the class II enzyme a triphosphate reductase. In the case of P. stutzeri, the class I enzyme is allosterically inhibited by dATP and the class II enzyme is stimulated. These different requirements make it difficult to understand how the reductases are regulated in vivo and how enzyme activity is attuned to the requirements of DNA replication. Further work with genetically modified bacteria is clearly required to answer some of these questions.

An additional, equally puzzling problem is how this situation has arisen during evolution. We suggested earlier that ribonucleotide reduction arose early during the evolution of life under anaerobic conditions during the transition of an RNA to a DNA world (20, 31). Of the enzymes present today, a class III-like reductase appeared to be the best candidate for an early enzyme. Class II and class I enzymes, in that order, evolved later, when oxygen appeared in the atmosphere. Oxygen became a major driving force for the diversification of ribonucleotide reductases, since the glycyl radical of class III enzymes is oxygen sensitive and new methods of radical generation were required during aerobiosis.

How do P. aeruginosa and other Pseudomonas species fit into this a model? The occurrence of all three classes in one bacterium could mean that each class, once it had evolved, was maintained. But why should an organism with a functioning class II enzyme evolve a class I reductase? One conceivable reason would be that the bacterium had lost the ability to synthesize B₁₂. However, P. denitrificans can make its own B₁₂ (8, 33), and also P. aeruginosa contains the necessary genomic information. A possible explanation may derive from the finding that the class I enzyme of P. aeruginosa is closely related to eukaryotic class I reductases and also possesses a high degree of sequence similarity to the enzyme of C. trachomatis, a bacterium containing many genes presumably transferred from its eukaryotic host (37). This finding suggests horizontal gene transfer as the source for the class I enzymes of pseudomonads. The question of why two active enzymes are maintained by the bacteria thus remains.

Before we can reach an understanding, more work is required, in particular concerning the division of labor between the two enzymes of P. aeruginosa during aerobic and microaerophilic growth. We also need to investigate reductases from other Pseudomonas species, particularly those that appear to contain only one of the two classes (Table 1).