Occurrence of Multiple Ribonucleotide Reductase Classes ing-ProteobacteriaSpecies

Eduard Torrents,1 Albert Jordan,1,* Margareta Karlsson,2 Isidre Gibert1

1Department de Gene`tica i de Microbiologia and Institut de Biologia Fonamental, Bacterial Molecular Genetics group, Universitat Auto`noma deBarcelona, 08193 Bellaterra, Spain2IT-Universitetet/KTH-Kista, Kista-fo¨rvaltningen, Electrum 214, 164 40 Kista, Sweden

Received: 8 May 2000 / Accepted: 13 June 2000

Abstract. Ribonucleotide reductase (RNR) is central to de novo synthesis of deoxyribonucleotides andessential for all living cells. Three classes have been described; class I is oxygen dependent andrepresented by two subclasses, Ia (NrdAB) and Ib (NrdEF); class II (NrdJ) is indifferent to oxygen; andclass III (NrdDG) is oxygen sensitive. More than one class can be found in an organism, reflecting theoxygen status of its environment. We have investigated, by using PCR and Southern blot, the occurrenceof the different classes among species of theg-Proteobacteria. Class III are present in all species tested,but the presence of the other classes varies. Some species contain one unique additional enzyme, classIa, Ib, or II, whereas others contain two additional enzymes, class Ia and Ib, or class Ia and II.

Pathogenic bacteria are becoming more and more toler-ant to standard antibiotic treatment [3]. Many enzymes inthe pathway leading to deoxyribonucleotide and DNAsynthesis are essential for the proliferating cell and are,therefore, looked upon as potential targets for antiprolif-erative drugs. Reduction of ribonucleotides to the corre-sponding deoxyribonucleotides by ribonucleotide reduc-tase (RNR) is the only de novo pathway for makingDNA building blocks, and, hence, this enzyme has beenused as a target in cancer therapies [12] and suggested asa target for antiviral and antiparasite therapies [5, 10]. Itwould thus be tempting to explore ribonucleotide reduc-tase as a target for new antibacterial drugs. However, onecomplicating factor is the presence of three differentclasses (I-II-III) of the enzyme. The ribonucleotide re-ductases show differences in cofactor requirement andgeneral architecture, but share similar modes of regula-tion and reaction mechanism [6]. Class I is oxygendependent and can be divided into two subclasses, Ia andIb (nrdABor nrdEF genes), class III is oxygen sensitive(nrdDG genes), and class II (nrdJ gene) functions under

both aerobic and anaerobic conditions. It has been shownthat many microorganisms have the ability to produceenzymes belonging to more than one class, whereasmulticellular eukaryotes depend on a single class (Ia).

New antibacterial therapy based on differences be-tween the microbial enzymes and their counterparts inthe eukaryotic hosts should have the potential to be bothselective and efficient. To be able to use RNR for thispurpose, it is essential to know which of the RNR genesare present in the pathogenic microorganism and whenthese different genes are expressed. The aim of this workwas to examine and compare the ribonucleotide reduc-tase genes present in a group of related bacteria by usingtwo different methods, Southern blot and PCR. We chosetheg-Proteobacteriasince many of them are pathogenicto humans, animals, or plants [1]. Furthermore, amongthese bacteria there are species known to have genes forall three classes, e.g.,Pseudomonas aeruginosa[9].

Materials and Methods

Bacterial strains, plasmids, growth conditions, and materials.Bac-terial strains and plasmids used in this work are listed in Tables 1 and3. The strains were grown normally at 37°C or 30°C in Luria-Bertanirich medium (LB) except for the photosynthetic bacteriumChromatiumvinosum, which was grown anaerobically with light at 25°C in minimalPfenning medium as described in [11]. Restriction endonucleases, T4DNA ligase, nucleotide mixture, and the hybridization reagents were

* Present address:Gladstone Institute of Virology and Immunology,University of California San Francisco, San Francisco, CA 94141-9100, USA.

Correspondence to:I. Gibert; e-mail: igibert@cc.uab.es

CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 346–351DOI: 10.1007/s002840010147 Current

MicrobiologyAn International Journal© Springer-Verlag New York Inc. 2000

obtained from Roche Molecular Biochemicals and used as specified bythe manufacturer. PCR primers were from SGS (Scandinavian GeneSynthesis, Ko¨ping, Sweden). Sequencing was performed by usingDYEnamic ET terminator cycle sequencing premix kit from AmershamPharmacia Biotech. Strains were kindly provided by the Coleccio´nEspan˜ola de Cultivos Tipo (CECT) except forC. vinosum, which wasprovided by the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ).

General molecular biology techniques.Plasmid and genomic DNAisolation, agarose gel electrophoresis, restriction endonuclease diges-tions, and DNA ligation were carried out essentially as described in[13]. Cloned PCR products were sequenced by using universal directand reverse primers (Amersham Pharmacia Biotech) and the specificPCR primers (Table 2).

Southern hybridizations. Genomic DNAs were digested withEcoRIexcept forAeromonas hydrophilaand Chromatium vinosum, whereBamHI was used. DNA fragments and DIG-labeledHindIII lambdamarker were separated by electrophoresis in 1% agarose gel, blotted toNylon-Membrane positively charged sheets (Roche Molecular Bio-chemicals) by using a high ionic strength buffer (203 SSC), and fixedto the sheets by UV irradiation (Fluo-Link FLX). Prehybridization andhybridization were performed at high (42°C) or low stringency (30°C)as described in [13]. All hybridization probes were generated byamplification with DNA Taq polymerase, by using universal primers(M13 direct and reverse) and plasmids carrying the different ribonu-cleotide reductase genes (Table 1). The PCR reaction was carried outas described below, except that the concentrations of dNTPs were 1 mM

of dATP, dGTP, and dCTP, 0.65 mM of dTTP, 0.35 mM digoxigenin-11-dUTP, and the program used: a) 2 min at 94°C, b) 30 cycles of 10 sat 94°C, 30 s at 57°C, and 3.5 min at 68°C, and c) 7 min at 68°C.Hybridized DNA was detected by using the alkaline phosphatase-conjugated anti-digoxigenin antibody from the DIG-DNA labelingdetection kit (Roche Molecular Biochemicals). For chemiluminescencedetection, the substrate used was CSPDt reagent, and the blots wereexposed to X-ray film (Bio Max ML, Kodak).

PCR amplifications. For PCR amplifications, sets of different oligo-nucleotide pairs were used designed to match conserved regions ofdifferent classes of ribonucleotide reductases. The oligonucleotide pairsand PCR conditions are shown in Table 2. In general, a 50-ml PCRreaction mixture contained 50 pmol of each primer, all dNTPs (at 0.2mM each), 5ml of 103 PCR buffer, and 1.5 U Taq polymerase(Amersham Pharmacia Biotech). Amplified products were purified withthe QIAquick PCR Purification Kit (Qiagen) and cloned in thepGEM-T (Promega) or pT7Blue T-Vector (Novagen) according to themanufacturers’ protocols, and transformed intoE. coli Nova blue cells(Novagen).

Results and Discussion

To investigate the distribution of ribonucleotide reduc-tase classes, we used two general methods, Southern blotand PCR. Both techniques are easily applied to serieswith many samples, and the conditions can be varied to

Table 1. Plasmids used in this work

Plasmid Description Reference

pUA446 pBluescript SK carrying a 3.2-kbp DNA fragment of theS. typhimurium nrdEFgenes, Apr. [7]pUA447 pBluescript SK carrying a 4.3-kbp DNA fragment of theS. typhimurium nrdABgenes Apr. [7]pIG051 pGEM-T carrying a 533-bp DNA fragment of theS. typhimurium nrdDgene, Apr This studypSQUIRE Lactobacillus leichmanniiclassIInrdJ gene, Apr. [2]pIG048 pGEM-T carrying a 729-bp DNA fragment of theP. aeruginosa nrdAgene, Apr [9]pIG049 pGEM-T carrying a 615-bp DNA fragment of theP. aeruginosa nrdJgene, Apr [9]pIG050 pGEM-T carrying a 1208-bp DNA fragment of theP. aeruginosa nrdDgene, Ap [9]

Table 2. Primers used for PCR amplifications

PrimerPair No. Sequence (59-39)

Peptidesequence Target PCR conditions

Expectedproduct (bp)

1 CC(AG)AC(CT)CGTCAGTTCAGCTCCTG PTRQFDSSCI nrdA Anneal: 55–60°Ca 676C(AT)ATCTCCAG(AG)CACAGGTTAGACTG QSNLCI L/CE(I)

2 CG(AT)TC(AC)TATACTCATATCAT(GT)CG RSYI THII/M(R) nrdB Anneal: 45–55°Ca 329TTGGC(AG)TT(AG)CCTTCCAT(CG)A(AT)TTC EL/IMEGNA(K)

3 GTGGCCGGC(AG)(AG)TGT(CG)CGCCGGTC VVIPDITF nrdE Anneal: 55–60°Ca 567CAG(AG)TAGCC(AG)TGCAG(AG)TTCATCTG QMNLHGYL

4 CTCCGATGGG(CT)TGCCG(CT)AG(CT)TTC T/SPMGCI RSF nrdD Anneal: 59–62°Ca 361TTCGTG(AG)AT(AG)CC(AG)ATGTA(AT)CC(CT)A(AG)(AC)G (S)LGYIGIHE

5 GGTCAGACGCCGTTTG GQTPFV nrdD Anneal: 45–55°Ca 369CACGCCGAGGTTGTTACG RNNLGV

All PCR reactions were run with the following program; a) 5 min at 94°C, b) 30 cycles of 1 min at 94°C, 1 min at the annealing temperatureas shown above, and 1 min1 5 s/cycle at 72°C, and c) 7 min at 72°C.Essential amino acids involved in catalysis are underlined.a Depending on the DNA template.

E. Torrents et al.: Ribonucleotide Reductase ing-ProteobacteriaSpecies 347

pick up signals from distantly related genes. We focusedon theg-Proteobacteriasince earlier studies have indi-cated a varied distribution of ribonucleotide reductaseclasses in members of this group [6].

Southern blot analysis.For class Ia and Ib we used theSalmonella typhimurium nrdABandnrdEF genes (Table1) as probes. For class II we used two different probesderived fromLactobacillus leichmanniiandPseudomo-nas aeruginosa. Both species have a class II ribonucle-otide reductase, but they differ in their amino acid se-quence [2, 9]. For class III we cloned and sequenced aS.typhimurium nrdDfragment by using primer pair num-ber 5 (Table 2; Accession Number: AJ250962).

A summary of the hybridization results obtainedunder high and low stringency conditions is given inTable 3. Under high-stringency conditions, we found thenrdAB, nrdEF, and nrdD genes in theEnterobacteri-aceaegroup with the exception ofProteus vulgarisandP. rettgeri, where only thenrdEF andnrdD genes weredetected. As in theEnterobacteriaceaegroup, thenrdAB,nrdEF, and nrdD genes are present inAcinetobacter

calcoaceticus, and in theVibrio genera only thenrdABandnrdD genes could be detected.

A weak signal corresponding to anrdEF band wasdetected inChromatium vinosum. No positive resultswere detected inAeromonas hydrophilaand P. aerugi-nosa. These negative results could be due to differencesin GC% composition between genomic DNA fromA.hydrophila (61%) and theSalmonellaprobes (53%).Therefore, we usedP. aeruginosaprobes with a higherGC content (64%) [9] and were able to detect class III inC. vinosum(64%) and class II and III inA. hydrophila.

PCR and nucleotide sequence analysis.PCR amplifi-cations were combined with sequencing to verify theamplicons and the results obtained in the Southern anal-ysis. Primer-pairs were designed for class Ia, class Ib,and class III from a comparison of amino acid andnucleotide sequences of known bacterial RNRs (data notshown). All PCR reactions were carried out as describedin Materials and Methods and in Table 2. A class Ia-specific fragment was detected inY. pestis, Y. pseudotu-berculosis, andY. enterocolitica, as well as inV. chol-

Table 3. Southern hybridization analysis

Species tested

Southern hybridization

Class Ia Class Ib Class II Class III

EnterobacteriaceaeEscherichia coliATCC 11775 11c 11c 2 11a,c

Salmonella typhimuriumATCC 15277 11c 11c 2 11a,b,c

Citrobacter freundiiATCC 8090 11 11 2 11Erwinia carotovoraATCC 15713 11a,b 11 2 11Serratia marcescensATCC 13880 11 11 2 11Enterobacter cloacaeATCC 13047 11 11 2 11a

Klebsiella pneumoniaeATCC 13883 11 11 2 11a

Morganella morganiiATCC 25830 11 1 2 11Yersinia pestisATCC 19428 11a,b,c 11a,b,c 2 1a,c

Yersinia pseudotuberculosisATCC 29833 11a,b 11a,b 2 1a

Yersinia enterocoliticaATCC 9610 1a,b 1 2 1a

Proteus vulgarisATCC 13315 2 1 2 1a

Proteus rettgeriATCC 29944 2 1 2 1a

Related generaVibrio choleraeATCC 14035 1a,c 2 2 1c

Vibrio parahaemolyticusATCC 17802 1 2 2 1Aeromonas hydrophilaATCC 7966 2d 2 1d 1d

OthersPseudomonas aeruginosaATCC 15692 1c,d 2 1c,d 1c,d

Acinetobacter calcoaceticusATCC 23055 11a,b 11a,b 2 11Chromatium vinosumDSM185 2 1a,b 2 1d

11 Positive hybridization at high stringency.1 Positive hybridization at low stringency.2 No hybridization detected at high or low stringency with bothSalmonellaandPseudomonasprobes.a Tested by PCR.b PCR fragment sequenced.c Sequence available in TIGR database.d Using aPseudomonas aeruginosaprobe.

348 CURRENT MICROBIOLOGY Vol. 41 (2000)

erae, E. carotovora, and A. calcoaceticus(Table 3).Class Ib was amplified fromY. pestis, Y. pseudotuber-culosis, C. vinosum, and A. calcoaceticus. The classIII-specific primers used were able to amplify a fragmentfrom E. coli, S. typhimurium, E. cloacae, K. pneumoniae,P. vulgaris, P. rettgeri, V. cholerae, and from the threeYersinia species. The results obtained are in completeagreement with the results from our Southern blot anal-ysis and also with available sequence data from TheInstitute of Genomic Research (TIGR) (Table 3). Tocheck the specificity of the PCR amplifications, wecloned some PCR fragments and sequenced them toverify their identity (accession numbers: AJ286844,AJ286845, AJ286846, AJ286847, AJ286848, AJ286849,AJ286850, AJ286851, AJ286852, AJ272153, AJ272154).Percentage of identity within the deduced NrdE proteinfragments ranged from 100% betweenY. pseudotuberculo-sisandY. pestis, to 69% betweenY. pestisandA. calcoace-ticus, and 60% betweenS. typhimuriumand C. vinosum,

which show the greatest degree of difference. The deducedNrdA protein fragments from this study show an identityclose to 95% when compared. Within the deduced NrdBprotein fragments the identity ranged from 100% to 89%.This can be contrasted with the low level of identity be-tweenE. coli andP. aeruginosaNrdB of 27% [9]. Class IIIRNR sequences from unfinished genomes of theg-Pro-teobacteriagroup (TIGR) show a high level of identity thatranges from 98% to 83%.

Ribonucleotide reductase occurrence.Can the classdistribution of ribonucleotide reductase be fitted into aphylogenetic framework provided by 16S rDNA com-parisons? In Fig. 1 the phylogenetic relationship betweenthe different species of this investigation based on their16S rDNA sequences are shown and compared with thedistribution of the different RNR classes. The close re-lationship within theEnterobacteriaceaeis evident, andthese bacteria also contain the same RNR classes, i.e.,

Fig. 1. Phylogenetic relationships among se-lectedg-Proteobacteriaand distribution ofribonucleotide reductase classes. The phyloge-netic tree was constructed from the available16S rDNA sequence data from the RibosomalDatabase Project (http://www.cme.msu.edu/RDP/html/analyses.html). *RNR sequence in-formation was obtained from the NCBI database (including unfinished microbial genomes).

E. Torrents et al.: Ribonucleotide Reductase ing-ProteobacteriaSpecies 349

class Ia, Ib, and III.P. vulgarisand P. rettgeri are theexception in this subgroup because, apart from class III,they contain only class Ib. The same classes as inEn-terobacteriaceaewere also found inA. calcoaceticus. Itis interesting to see that this bacterium, described asaerobic, contains the genes for the oxygen-sensitive ver-sion of the enzyme. Like theProteusgenera,C. vinosumand theVibrio sp. were found to have only one subclassof class I, together with class III. Since class Ib can befound in Gram-positive bacteria and class Ia is alsofound outsideEnterobacteriaceae[6], the most likelyevolutionary scenario for RNR class I is a gene duplica-tion that occurred prior to the divergence of the Eubac-teria, followed by selective loss of one or the othersubclass.

What could be the role of class Ib inE. coliand otherbacteria where the active oxygen-dependent ribonucle-otide reductase is class Ia? One interesting observationthat might shed some light on this question is that inLactococcus lactisclass Ib has been found to compensatefor a class III mutation during standard laboratory anaer-obic conditions [8]. The recently published structure ofthe class Ib small subunit provides a possible clue to thisobservation [4]. The NrdF protomer contains a narrowchannel that could permit easier access for oxygen to theferrous iron site. This would allow the NrdF protein toefficiently generate the tyrosyl radical, essential for ca-talysis, even at low oxygen levels. We suggest that thisadditional aerobic enzyme could function under mi-croaerophilic conditions where the oxygen availability istoo low to activate class Ia but high enough to inactivateclass III.

Class II is a versatile enzyme, not affected by thepresence or absence of oxygen. Hence, it is not surpris-ing that it is found in all forms of life, Archaeabacteria,Eubacteria, and in at least one eukaryote,Euglena gra-cilis [6]. However, its presence in theg-Proteobacteriaseems scarce, and apart fromP. aeruginosa[9], we couldfind it only in A. hydrophila. Whether this is a result ofgene loss or an example of horizontal gene transfer is anopen question. The presence of class II is an anomaly inour RNR distribution results and does not fit well in theg-Proteobacteriagroup, where it is represented in onlytwo species. Finally, the only common denominator forall tested species is the presence of class III RNR, theoxygen-sensitive version of the enzyme. This is notsurprising since most of the bacteria tested are describedas facultative anaerobes, and also, this is possibly themore ancient version of all RNRs [14].

Taken together, our results imply that phylogenyalone can not be used to anticipate which classes ofribonucleotide reductase are present in a bacterial spe-cies. Also, protein phylogenies can be problematic be-

cause of biases in species, lateral gene transfer, geneduplications, and artifacts of phylogenetic reconstructionmethods. But considering phylogeny can still be helpfulwhen evaluating results from methods like Southern blotand PCR. A more difficult issue, and a very importantone when considering RNR as a target for antibacterialdrugs, is to determine under what conditions the differentclasses are active.

The presence of multiple RNRs with distinct re-quirements in a microorganism makes targeted therapyless straightforward than originally anticipated, despitethe possibility of utilizing differences between the hostenzyme and those of the pathogen. This calls for a broadapproach to the study of DNA precursor synthesis inpathogens.

ACKNOWLEDGMENTS

We thank Solveig Hahne for help with the sequencing of PCR-productsand Patrick Young for constructive criticism of the manuscript. We alsothank Dr. Å. Forsberg and Dr. R. Andersson for providing differentYersiniaand Vibrio chromosomal DNA, respectively. Our work wassupported by a grant from the Spanish Direccio´n General de Ensen˜anzaSuperior e Investigacio´n Cientıfica (PB97-0196) and partially by Fun-dacioMaria Francesca de Roviralta, Spain to I. Gibert.

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