Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria

Promoter identification and expression analysis ofSalmonella typhimurium and Escherichia coli nrdEFoperons encoding one of two class I ribonucleotidereductases present in both bacteria

Albert Jordan, Eugeni Aragall, Isidre Gibert and JordiBarbe *Department of Genetics and Microbiology, Faculty ofSciences, Autonomous University of Barcelona,Bellaterra, 08193, Barcelona, Spain.

Summary

Salmonella typhimurium and Escherichia coli cellshave two different class I ribonucleotide reductasesencoded by the nrdEF and nrdAB operons. Despitethe presence of one additional ribonucleotide reduc-tase, the nrdAB -encoded enzyme is essential to theaerobic growth of the cell because nrdAB -defectivemutants of both species are not viable in the presenceof oxygen. Several factors controlling nrdAB genetranscription have been analysed intensively. Nothingis known about the expression of the nrdEF genes. Tostudy this subject, and after cloning of E. coli nrdEFgenes and sequencing of their 5 ' ends, the promoterof this operon has been identified by primer extensionin both bacterial species. The � 1 position was 691 bpand 692 bp upstream of the translational start pointsof the nrdE genes of S. typhimurium and E. coli ,respectively. Downstream of the �1 position, andbefore the nrdE gene, two open reading frames(ORFs) of 81 and 136 amino acid residues are presentin both bacteria. The synthesis of a polypeptide with amolecular mass of 9 kDa, corresponding to the first ofthese two ORFs, was observed by using the T7 RNApolymerase expression system. Comparison of theamino acid predicted sequence of this ORF reveals asignificant similarity with glutaredoxin proteins. Com-petitive, reverse-transcription polymerase chain reac-tion experiments indicate that transcription from thenrdEF promoter normally takes place in wild-typecells. nrdEF transcription is increased by hydro-xyurea, which inhibits class I ribonucleotide reduc-tase activity, in both RecA � and RecA ÿ cells. nrdA ts

mutants show a higher level of nrdEF transcription

than wild-type cells at either the permissive or therestrictive temperature. nrdEF expression was unaf-fected by changes in DNA supercoiling whethercaused by the introduction of either topA ::Tn 10 andhns ::Tn 10 mutations or by the inhibition of DNA gyr-ase with the antibiotic novobiocin. In contrast to thenrdAB genes, the nrdEF operon is not essential tothe cells because nrdEF -defective mutants are viableunder both aerobic and anaerobic conditions.

Introduction

Deoxyribonucleotides required for DNA synthesis areformed de novo by the enzyme ribonucleotide reductase(RR). Three classes of RR are known to date (Reichard,1993a). The first of them, class I, contains aerobicenzymes, which are present in higher organisms and incertain prokaryotes. Escherichia coli ribonucleoside diphos-phate reductase (RDP reductase) is the best known ofthis kind of enzyme. In this bacterium, the nrdA andnrdB genes encode the a and b polypeptidic chains,which form the R1 (a2) and R2 (b2) subunits of the RDPreductase. These two genes are transcribed as a 3.2 kbpolycistronic mRNA (Fuchs and Karlstrom, 1976; Hankeand Fuchs, 1983). A free radical located in Tyr-122 andan oxygen-linked dinuclear iron centre are essential com-ponents of the catalytic process carried out by this enzyme(Larson and Sjoberg, 1986). Class II enzymes, present inmany microorganisms, use adenosylcobalamin as a radi-cal generator (Blakley et al., 1966). With respect to theclass III, the best known so far is the anaerobic ribonucleo-tide triphosphate reductase (RTP reductase) of E. coli,encoded by the nrdD gene (Reichard, 1993b). It containsan iron–sulphur centre and forms a glycyl radical with S-adenosine methionine as cofactor.

As class II enzymes can function in both aerobic andanaerobic conditions, organisms containing this kind ofenzyme do not seem to require any additional RR. Thereare two explanations as to why E. coli cells, which do notpresent class II enzymes, have genes encoding classes Iand III RR. The first is the fact that E. coli is a facultativeanaerobic microorganism. The second is the oxygendependence of class I RR (Fontecave et al., 1992). The

Molecular Microbiology (1996) 19(4), 777–790

# 1996 Blackwell Science Ltd

Received 26 July, 1995; revised 4 October, 1995; accepted 6October, 1995. *For correspondence. E-mail [email protected]; Tel.(343) 5811837; Fax (343) 5812387.

g

E. coli nrdAB genes are considered as essential genesbecause only conditional-lethal nrdAB mutants may beobtained in the presence of oxygen (Fuchs et al., 1972;Hantke, 1988). Nevertheless, and surprisingly, Salmo-nella typhimurium and E. coli have a second RR of classI (Jordan et al., 1994a). This new RR, constituted by theR1E and R2F proteins which are homodimers of thenrdE and nrdF products, has a limited identity with otherclass I enzymes although it contains many of their cataly-tically important residues. Thus, the S. typhimurium R1E/R2F enzyme in vitro catalyses the reduction of CDP, withthe glutaredoxin system as the hydrogen donor, and con-tains an oxygen-linked dinuclear iron centre and a tyrosylradical (Jordan et al., 1994b). In this case, the reductionreaction is strongly stimulated by dATP. Together thesefindings support the definition of a separate subgroup(Ib), different from the ‘classical’ class Ia, to which theR1E/R2F enzyme should belong.

The significance of this new enzyme in vivo as well asthe control of its expression are still unknown. However,it is clear that nrdEF genes only suppress an nrdAB-defec-tive phenotype when an additional copy is present in eitherF' or cloning plasmids (Jordan et al., 1994a), suggestingthat the chromosomal locus is not expressed. Here, wedemonstrate that nrdEF genes are expressed under nor-mal culture conditions, and that nrdEF transcription isincreased in nrdAts mutants, but apparently not enoughto allow complementation of this mutation until a secondcopy of the nrdEF operon is placed in the chromosome.Our previous results had established that a fragment ofabout 800 bp located upstream of the S. typhimuriumnrdE gene was entirely necessary for normal transcriptionof these genes, indicating that the nrdEF promoter mustbe located in this region (Jordan et al., 1994a). In the pre-sent work we have also determined the transcriptionalstart points of the S. typhimurium and E. coli nrdEF oper-

ons. Expression of these genes under several conditionsis also analysed.

Results

Isolation of the E. coli nrdEF genes and sequencingof its 5 '-end region

Prior to analysis of E. coli and S. typhimurium nrdEFexpression, it was necessary to isolate and sequencethe E. coli nrdEF promoter. To achieve this objective,and taking advantage of the fact that nrdEF genes arelocated upstream of the proU operon in unit 57 of boththe S. typhimurium and the E. coli chromosome (Jordanet al., 1994a), we identified the clone of the E. coli Koharalibrary containing the nrdEF operon (Kohara et al., 1987).EMBL4 clone 8G10(445) of this library was used as asource of DNA. Southern analysis (with a 2.7 kb ClaI–Hin-dIII fragment harbouring the S. typhimurium nrdE gene) ofDNA obtained from these l phage particles grown overstrain W3110 revealed that the E. coli nrdEF genes werecontained in an 8 kb ClaI fragment, which was cloned inthe pBSK(+) vector, giving rise to plasmid pUA426. Thisplasmid was able to abolish both the temperature depen-dence and the hydroxyurea hypersensitivity of the nrdAts

nrdB1 mutant KK450 of E. coli, showing that the nrdEFgenes of this bacterium were present and fully functional.Complementation assays with 5' deletions of the 8 kb ClaIfragment indicated that, as happens in S. typhimurium, afragment of about 4 kb is required to support the growthof the KK450 strain at 428C (Fig. 1A). Further Southernand restriction analyses showed that, in agreement withdata previously obtained for S. typhimurium nrdEF genes,the E. coli nrdEF promoter must be present in a 1.5 kbEcoRI–AvaI fragment. This fragment was cloned in thepBSK(+) plasmid, to give pUA523, and then sequenced.

Figure 1 shows the 5'-end of the E. coli nrdEF operon

# 1996 Blackwell Science Ltd, Molecular Microbiology, 19, 777–790

Fig. 1. The E. coli nrdEF operon and its promoter region.A. Schematic representation of the nrdEF operon and its surrounding region contained in the chromosomal fragment carried by plasmidpUA426. Only relevant restriction sites are given. The ability of inserts present in pUA426, pUA425 and pUA524 plasmids to complement thenrdAts mutation of strain KK450 is indicated. The lower section represents the DNA region sequenced either in this work (plasmid pUA523) orby Gowrishankar (1989). The promoter and the transcriptional starting point are indicated by the letter P. The percentages of identity of bothnucleotide and deduced amino acid sequences between E. coli and S. typhimurium (Jordan et al., 1994a) are indicated for promoter regionand genes.B. Nucleotide sequence of the E. coli nrdE gene upstream region. The 1144 nucleotides of the sequenced fragment of the E. coli nrdEupstream region which are common to those of S. typhimurium are presented. The deduced amino acid sequences of ORF1 and nrdE ' areindicated below the nucleotide sequences. The cysteines of the CXXC active centre of the hypothetical electron donor encoded by ORF1 areencircled. Nucleotide and amino acid sequences from S. typhimurium are shown above and below, respectively, only when they differ from E.coli sequences. , and symbols indicate nucleotides that are added or deleted in S. typhimurium, respectively. The predicted 710 and 735sequences for the nrdEF promoter, as well as a putative DnaA box and the Shine–Dalgarno (SD) sequences present upstream of ORF1 andnrdE, are underlined. An arrowed dot indicates the transcriptional starting site (+1) determined by primer extension. Several translationalinitiation and stop codons are boxed. A consensus sequence found in the nrdAB and nrdEF promoters of both E. coli and S. typhimuriumregions is also boxed close to the 710 region. Complementary sequences to the oligonucleotides used for primer extension are underlinedand labelled Ec1 for E. coli and St1 to St4 for S. typhimurium. Corresponding or complementary sequences to oligonucleotides RTup andRTlow, respectively, used for RT-PCR, are overlined and labelled. AccI restriction sites used for construction of the RT-PCR competitormolecule are also indicated.

778 A. Jordan, E. Aragall, I. Gibert and J. Barbe


nrdEF operon expression 779

sequenced in this work, in comparison to the S. typhimur-ium sequence (Jordan et al., 1994a), as well as a sche-matic representation of the previously reported sequenceof the 3'-end of the E. coli nrdF gene (Gowrishankar,1989). Upstream of both the E. coli and the S. typhimuriumnrdE genes, two open reading frames (ORFs) are present.The degree of identity between these two ORF from bothbacteria is very high (80%). The sequenced fragments ofE. coli nrdE and nrdF genes show high identity with thoseof S. typhimurium (71% and 78%, respectively) (Fig. 1A).Oddly, the translational start codon of the E. coli nrdEgene seems to be the UUG triplet, as in S. typhimurium(Jordan et al., 1994a). A canonical TATA box precededby a 735 promoter consensus sequence is absent imme-diately upstream of the E. coli nrdE gene.

Quantification of the nrdEF operon expression

The fact that mutants in the nrdA gene of both E. coli andS. typhimurium are conditionally lethal suggested thatnrdEF genes might not be expressed in cells of both bac-teria. When it is suspected that a given gene is very weaklyexpressed, several reverse transcription/polymerasechainreaction (RT-PCR)-derivative techniques, which enhancethe sensitivity for detection of transcripts compared tostandard Northern analysis by 1000–10 000-fold(Mocharla et al., 1990), may be employed. In our case,and since we were unable to detect the nrdEF mRNA byNorthern experiments in both E. coli and S. typhimuriumcells, we used a specific, competitive PCR-amplificationmethod in order to identify and compare relative levels ofexpression between samples.

Results obtained indicated that nrdEF operon transcrip-tion takes place in MC1061 E. coli wild-type cells (Fig. 2).The highest level of nrdEF mRNA was observed in E. colicells carrying either the multicopy plasmid pUA524containing the nrdEF operon or a second copy of thenrdEF genes in their chromosome as a consequence ofthe insertion of a mini-Tn5-nrdEF transposon (UA6047).Moreover, and unexpectedly, nrdEF transcription notonly occurs in cells of the nrdAts KK450 mutant, but it ishigher than in wild-type E. coli cells. The mini-Tn5-nrdEFinserted in the chromosome of strain KK450 (UA6069)dramatically increases the nrdEF mRNA level (Fig. 2).This agrees with the fact that the presence of this transpo-son eliminates the temperature sensitivity in that mutant.Hydroxyurea, which is a strong inhibitor of the activity ofboth nrdAB- and nrdEF-encoded ribonucleotide reduc-tases (Sinha and Snustad, 1972; Jordan et al., 1994b)and stimulates nrdAB transcription (Gibert et al., 1990),increased nrdEF expression in all cases. Wild-type andnrdAts S. typhimurium cells presented the same behaviouras that obtained with their respective E. coli strains (data

not shown).Treatment with hydroxyurea enabled us to detect nrdEF

expression in both E. coli and S. typhimurium cells byNorthern hybridization using nrdEF mRNA specific ribo-probes (data not shown). Results obtained showed thatthe nrdEF mRNA synthesized after hydroxyurea treatmentis about 4 kb long. This value is consistent with the theo-retical size of the nrdEF operon calculated from thesequencing data.

Behaviour of chromosomal nrdEF mutants

By marker exchange with a suicide plasmid (pUA474) con-taining a spectinomycin-resistance (SpcR) cassette inser-ted in the nrdE gene (see the Experimental procedures)we obtained nrdEF-defective clones (Fig. 3). Theseresults demonstrate that nrdEF genes are not essentialto cell viability. No phenotypic differences were detectedbetween wild-type and nrdE ::OSmR/SpcR (SmR = strepto-mycin resistance) cells in either the presence or theabsence of oxygen. On the contrary, the viability ofnrdABts cells containing the mini-Tn5-nrdEF transposonat the restrictive temperature is decreased when eitherof the two nrdEF operons is mutated (Fig. 4), indicatingthat both copies are necessary to enable growth at 408C.Furthermore, KK450 nrdEF ::OSmR/SpcR cells are moresensitive to the temperature than KK450 cells (Fig. 4).The fact that nrdEF transcription is practically the samein both wild-type and nrdE ::OSmR/SpcR cells (Fig. 2) indi-cates that nrdEF genes are not autoregulated.

Transcriptional start points of the nrdEF genes ofS. typhimurium and E. coli

Previous work had suggested that the promoter of E. coliand S. typhimurium nrdEF genes should be at the begin-ning of their 800 bp upstream region, because almost allof this fragment is required for the nrdEF operon to beable to complement any nrdA-conditional mutation (Jordanet al., 1994a). This hypothesis is also supported by thesize of the nrdEF mRNA obtained in Northern experimentscited above. Furthermore, a clear promoter cannot beidentified by sequence similarity to the s

70 consensussequence in the DNA region immediately upstream ofthese genes.

To map the promoter, primer extension was performedin strains of E. coli and S. typhimurium with and without aplasmid carrying its respective nrdEF operon in theabsence or presence of hydroxyurea. The main signalobtained after screening with several oligonucleotides(see the Experimental procedures) is an A nucleotidelocated 66 (S. typhimurium ) and 67 (E. coli ) base pairsupstream of the translational start of the first ORF(Fig. 5). The same transcription start site was identified



irrespective of the species, the presence of multiple copiesof nrdEF, or growth in hydroxyurea (Fig. 5). The 710region of this promoter, TAGTAT, conforms quite wellwith the consensus Pribnow box. The corresponding735 region is predicted to be TTGAAT (Fig. 1B).

There are two DnaA boxes close to the +1 position ofnrdAB genes of both E. coli and S. typhimurium (Augustinet al., 1994; Jordan et al., 1995). A putative DnaA box,identified by homology to the established consensussequence (Schaefer and Messer, 1991), is present 89 bpand 91 bp upstream of the +1 position of nrdEF operonsof E. coli and S. typhimurium, respectively (Fig. 1B). Noother canonical regulatory sequence was found in nrdEF

promoters.

Expression of ORF1 and ORF2 contained in thenrdEF operon

The region between the promoter and the nrdE genecontains two ORFs encoding hypothetical proteins of 81and 136 amino acid residues, with calculated molecularmasses of 9123 Da and 15 234 Da, respectively (Fig. 1A).Thus, the nrdEF operon appears to be composed of fourgenes: ORF1, ORF2, nrdE, and nrdF. The function ofthe products of nrdE and nrdF is known. Nevertheless,


Fig. 2. Relative transcription of nrdEF genes in several E. coli strains. Total RNA from exponentially growing cells was obtained and analysedby the semi-quantitative competitive RT-PCR assay as described in the Experimental procedures. A constant amount of competitor wasadded to PCR reactions containing the experimental cDNA samples. Following amplification, 30% portions of the PCR products were resolvedon a 3% agarose/EtBr gel (upper panels). fX174/Hinf I DNA was used as the molecular weight marker (MW). The ratio of amplified target tocompetitor PCR products is shown for each sample (lower panels).A. Relative expression in the wild type (lanes 1 and 5), in nrdE ::OSmR/SpcR (lane 2), nrdAts (lane 3), and wild-type cells harbouring plasmidpUA524 carrying the nrdEF operon (lanes 4 and 6) growing in the absence (lanes 1 to 4) or in the presence (lanes 5 and 6) of hydroxyurea at50 mM. With the exception of lane 6, where a 10-fold dilution of the competitor was necessary, the same amount of competitor was added forall samples.B. In an independent experiment, the relative expression of the E. coli nrdAts mutant containing an additional copy of the mini-Tn5-nrdEFtransposon inserted in its chromosome (UA6069 strain) was determined (lane 3) in comparison to the values obtained in the same experimentfor wild-type (lane 1) and nrdAts (lane 2) cells.


there was no evidence that ORF1 and ORF2 might betranslated; therefore, we analysed the expression of thenrdEF operon in a bacteriophage T7 RNA polymeraseexpression system. To perform this, the pBSK derivatives,plasmids pUA338 and pUA539, carrying either the wholeS. typhimurium nrdEF operon or only ORF1 and ORF2downstream of the bacteriophage T7 promoter, respec-tively, were introduced into the E. coli strain DH5(pGP1-2), which harbours the T7 polymerase gene under thecontrol of the bacteriophage l promoter and a tempera-ture-sensitive repressor (Tabor and Richardson, 1985).Upon heat-induction of the T7 RNA polymerase gene,plasmid-encoded proteins were selectively labelled with[35S]-methionine and separated on an SDS–polyacryl-amide gel, as described in the Experimental procedures.A polypeptide with a molecular mass of 9 kDa was ob-served in lysates of strains carrying the plasmids men-tioned above but was absent from lysates of strains carry-ing the vector alone (Fig. 6). The size of this polypeptideconforms with the predicted size of ORF1. The sameresult was obtained when the expression of the E. coliORF1 contained on pUA524 and pUA537 plasmids wasanalysed (data not shown). The ORF2 product, with anexpected molecular mass of 15 000 Da, was not detectedin either bacterium when this system of expression wasused, although nrdE and nrdF gene products were clearlyobtained under the same experimental conditions

(Fig. 6). These results conform with the presence of aputative ribosome-binding site upstream of ORF1 but notof ORF2 (Fig. 1B).

Analysis of nrdEF operon expression by using lacZfusions

To further analyse the expression of the nrdEF operon ofS. typhimurium, a fusion between lacZ and these geneswas constructed. The promoter and the 5'-coding regionof nrdE were cloned in plasmid pUJ8 (de Lorenzo et al.,1990) upstream of the promoterless trp'–'lacZ region, giv-ing rise to plasmid pUA451. Afterwards, a Not I fragment ofthis plasmid containing the nrdE ::lacZ fusion was insertedinto the only Not I cloning site of the pUTmini-Tn5Km(de Lorenzo et al., 1990) to give pUA454. This mini-Tn5-nrdE ::lacZ plasmid fusion was transferred by conjugationto a rifampicin-resistant (Rif R) mutant of S. typhimuriumTR2279 (UA1573 strain). The presence of the desiredfusion in the chromosome of 12 independent exconjugantswas established by both Southern blot analysis and b-galactosidase production (data not shown). This proce-dure confirmed the unequal distribution of all the fusionsaround the chromosome in the various clones. BecauseTR2279 is a RecA7 derivative, the nrdE ::lacZ fusions pre-sent in its chromosome were afterwards transferred byP22-mediated transduction into a RecA+ background(LT2 strain). One of these transductants, showing a typicalb-galactosidase basal level (UA1740 strain), was selectedfor use.


Fig. 3. Southern analysis of DraI- and SacII-digested chromosomalDNA from the MC1061 E. coli strain (lane 1), and its nrdE ::OStrR/SpcR derivative clone (UA6055; lane 2) obtained by markerexchange as described in the text. The DNA probe used was a3 kb DraI–SacII fragment from plasmid pUA446 containing the E.coli nrdE gene. The integration of the O cassette, carrying a DraIrestriction site in each of its repetitive ends, produces the loss ofthe 3 kb hybridization band originating two new bands with sizes of1.7 and 1.3 kb. A HindIII digest of digoxigenin-labelled lambda DNAused as a molecular weight marker is shown in lane 3. Numbers onthe right are sizes in kilobases.

Fig. 4. Effect of temperature on the relative plating efficiency ofnrdAts (KK450), nrdAts nrdE ::OSmR/SpcR (UA6086), nrdAts mini-Tn5Km-nrdEF (UA6069), nrdAts nrdE ::OSmR/SpcR mini-Tn5Km-nrdEF (UA6087) and nrdAts mini-Tn5Km-nrdE ::OSmR/SpcR

(UA6068) cells. For each strain, the relative plating efficiency is theratio between colonies obtained at 408C and 308C. Platingefficiency of strain UA6086 was lower than 1076.


Hydroxyurea is a strong inducer of the nrdEF operon(Fig. 2). This compound has also been proven to triggerthe SOS system (Barbe et al., 1987). To determine if thehydroxyurea-mediated induction of nrdEF is SOS related,the behaviour of the S. typhimurium nrdE ::lacZ fusion inhydroxyurea-treated RecA+ and RecA7 strains was ana-lysed. Figure 7 shows that this compound increases thenrdE ::lacZ transcription in both RecA+ and RecA7 cells,indicating that its effect is not SOS dependent.

Expression of nrdAB genes in E. coli has been shown tobe triggered by treatments which inhibit DNA replication,such as mitomycin C, thymine starvation in Thy7 mutants,and incubation at the restrictive temperature of severaldnats mutants (Filpula and Fuchs, 1977; 1978; Gibertet al., 1990). Nevertheless, neither mitomycin C (Fig. 7)nor thymine starvation (data not shown) induced nrdEFtranscription. Moreover, S. typhimurium nrdAts and dnaAts

mutants did not increase nrdEF expression when shiftedfrom 308C to 428C (data not shown).

The expression of the proU operon of both E. coli andS. typhimurium is under supercoiling regulation and as aconsequence is sensitive to the action of hns- and topA-encoded proteins (Owen-Hughes et al., 1992; Hinton etal., 1992). The effect of supercoiling in the expression ofsome genes does not seem to be related to the chromo-somal domain in which they are located (Pavitt et al.,1993) but rather with their adjacent regions (Chen et al.,1992). Because of the poor expression of the nrdEFoperon when located in its natural position in the chromo-some of the wild-type cells, and since these genes are

upstream of the proU operon, we decided to study nrdEFtranscription in both hns ::Tn10 and topA ::Tn10 mutantsto analyse the hypothetical influence of the supercoilingdegree. Data obtained with five clones containingindependent transpositional insertions of the nrdE ::lacZfusion indicate that neither the presence of these muta-tions nor the addition of novobiocin, an inhibitor of theDNA gyrase enzyme, appreciably affected the expression


Fig. 5. Transcription start mapping for the S. typhimurium (A) and E. coli (B) nrdEF operons by primer extension analysis. Lanes T, C, G,and A show products of sequencing reactions of pUA338 (A) and pUA524 (B) plasmids carried out with the same primers used for primerextension. Only the relevant DNA sequence is shown. The nucleotide corresponding to the transcription start site is indicated by an arrow.See the Experimental procedures for further details.A. Transcription start site of the S. typhimurium operon. Primer extension and sequencing reactions were performed using oligonucleotideSt3. Total RNA was extracted from LT2 cells carrying plasmid pUA338 (lane 1), LT2 cells (lanes 2 and 3), and UA1740 cells (lanes 4 and 5),and growing in the presence (lanes 3 and 5) or in the absence (lanes 1, 2, and 4) of hydroxyurea at 50 mM. The same results were obtainedwith primer St4.B. Transcription start site of the E. coli operon. Primer extension and sequencing reactions were carried out using oligonucleotide Ec1. TotalRNA was extracted from MC1061 cells (lanes 1 and 2) and DH5aF' cells carrying plasmid pUA524 (lanes 3 and 4) and growing in thepresence (lanes 2 and 4) or in the absence (lanes 1 and 3) of hydroxyurea at 50 mM.

Fig. 6. Expresion of the nrdEF operon. Polypeptides encoded byvarious pBSK-derived plasmids containing the S. typhimuriumnrdEF operon downstream from a bacteriophage T7 promoter werelabelled as described in the text and separated by SDS–PAGE.Lanes: 1, pBSK (vector); 2, pUA539 (harbouring ORF1, ORF2 andthe 5'-end of S. typhimurium nrdE ); 3, pUA338 (containing thewhole S. typhimurium nrdEF operon). Positions of nrdE, nrdF andORF1 products are indicated, as well as the molecular massstandards (in kDa) run in parallel and stained with Coomassie blue.


of nrdEF genes in S. typhimurium, regardless of the posi-tion of the fusion in the chromosome (data not shown). Thebehaviour of the E. coli nrdEF operon, also analysed byusing a chromosomal nrdE ::lacZ fusion constructed asdescribed in the Experimental procedures, was thesame as that of S. typhimurium under all conditions tested(data not shown).

Discussion

The fact that the presence of a second ribonucleotidereductase in both S. typhimurium and E. coli cells didnot prevent the isolation of several independent lethal-conditional mutants in both species had been initiallyexplained by the hypothesis that under normal circum-stances the chromosomal nrdEF genes were silent (Jor-dan et al., 1994a). Nevertheless, results presented inthis work clearly show that transcription of nrdEF genesdoes occur. Furthermore, nrdAts mutants present higherexpression of the nrdEF operon than the wild-type strains.Why, then, is the inactivation of the nrdAB operon lethal toS. typhimurium and E. coli cells? The easiest answer tothis question could be that the R1E/R2F enzyme is not pro-duced in sufficient quantities to supply the minimal dNTPconcentration required to support DNA replication. Theabolition of temperature sensitivity in nrdAts mutants bythe presence of a second copy of the nrdEF operon intheir chromosomes should be due to the higher enzymelevel which these cells present as a consequence of theincrease in the nrdEF transcription detected (Fig. 2). Inagreement with this possibility, inactivation of one of the

two copies of the nrdEF operon in the KK450 mini-Tn5-nrdEF cells (UA6069 strain) dramatically decreased cellviability at 408C (Fig. 4). The low level of R1E/R2Fenzyme might also be due to post-transcriptional controlat either the mRNA or protein level, although the nrdEFoperon does not contain secondary structures resemblingattenuators. Furthermore, translation of nrdEF mRNAmight be low because the nrdE start codon is TTG.

Another explanation for the nrdAts phenotype could bethat the R1/R2 and R1E/R2F-ribonucleotide reductionsystems have a common component, and that the affinityof the nrdAB enzyme for this hypothethical element wasthe highest. In this respect, little is known about the com-pounds involved in the R1E/R2F-mediated ribonucleotidereduction in vivo. To date, it has been demonstrated that,in vitro, R1E/R2F reductase reduces CDP to dCDP withpractically the same efficiency as the R1/R2 reductase.DTT and reduced glutaredoxin are used as hydrogendonors by both enzymes (Jordan et al., 1994b). R1/R2and R1E/R2F systems are different in that the formerdoes not use reduced thioredoxin as the hydrogendonor. The dATP is the best allosteric activator of R1E/R2F (Jordan et al., 1994b), whereas it is a strong inhibitorof the R1/R2 reductase (Reichard, 1993a). The predictedamino acid sequence encoded by the ORF1 upstream ofthe nrdE gene shows a significant level of identity with sev-eral similarly sized glutaredoxin sequences, including thetypical CXXC motif of the active centre (Fig. 1B) (Hooget al., 1983). These data suggest that the product ofORF1 could be a specific electron donor for the R1E/R2F enzyme.


Fig. 7. Induction of the nrdEF operon,measured as b-galactosidase production, inwild-type (UA1740) and RecA7 (UA1739)cells of S. typhimurium carrying an nrdE ::lacZfusion in the absence and in the presence ofeither hydroxyurea at 50 mM or mytomycin Cat 20mg ml71.


Recently, and after enzyme purification from cellextracts and PCR-primer design, the gene encoding thelarge subunit of the ribonucleotide reductase from Myco-bacterium tuberculosis has been isolated and sequenced(Yang et al., 1994). Sequence comparisons indicate thatthis gene corresponds to the nrdEF subclass. Thus, inthis bacterium, the nrdEF reductase seems to show nor-mal function in vivo. This particular nrdE gene is not imme-diately followed by an nrdF gene. Surprisingly, in view ofthe fact that M. tuberculosis and S. typhimurium are notphylogenetically close, their nrdE products show a highdegree of identity (71%). This fact could indicate that therole of nrdEF genes is highly conserved in the bacterialworld, although the nrdEF system cannot be essential inE. coli and S. typhimurium cells because nrdEF knockoutmutants are viable. It is worth noting that there is moreidentity between the DNA sequences of ORF1 in E. coliand S. typhimurium than between those of their nrdEand nrdF genes (Fig. 1), although the closest identity isbetween the nrdAB genes of both enterobacteria (87%)(Jordan et al., 1995). The higher variability of the S. typhi-murium and E. coli nrdEF genes in comparison with that ofnrdAB genes would be in accordance with the main rolethat the R1/R2 enzyme seems to play in both bacteria.Nevertheless, the homology between the promoters ofthe nrdEF operon of both enterobacteria (80.5%) ismuch higher than that shown by that of nrdAB genes (Jor-dan et al., 1995). Nothing is known about the possible pre-sence of an nrdAB-like operon in M. tuberculosis.Moreover, the sequence of a fragment of an nrdA-likegene of Mycobacterium leprae (corresponding to residues433–482 of its homologous R1 protein of S. typhimurium )has been determined and investigated for its significantidentity with the human R1 (Hartskeerl et al., 1990). Theidentity between this nrdA fragment of M. leprae and S.typhimurium is dramatically lower (27%) than that shownby the M. tuberculosis and S. typhimurium nrdE genes.

Transcription of E. coli nrdAB genes has been shown tobe cell-cycle dependent and stimulated by Fis and DnaAproteins (Sun and Fuchs, 1992; Augustin et al., 1994).Nevertheless, there is no relationship between those pro-teins and the cell-cycle regulation of nrdAB (Sun et al.,1994). However, E. coli dnaAts mutants increased nrdABtranscription when shifted to the restrictive temperature(Filpula and Fuchs, 1978). The fact that nrdEF transcrip-tion was not increased in E. coli dnaAts mutants at 428Csuggests that nrdAB and nrdEF operons may present adifferent overall regulation despite the presence upstreamof nrdEF of a canonical DnaA box. Moreover, nrdAB andnrdEF expression is stimulated by hydroxyurea. Thisincrease might be an indirect consequence of dNTP deple-tion resulting from the inhibition of ribonucleotide reduc-tase activity. This hypothesis would agree with the higherlevel of nrdEF expression in the nrdAts KK450 strain at

308C, because the activity of the R1/R2 enzyme of thismutant is lower than that of the wild type even at the per-missive temperature (Fuchs et al., 1972). However, theexistence of some partial common controls betweenboth operons is a possibility that must not be dismissed.Thus, the promoter regions of nrdAB and nrdEF operonspresent the consensus sequence CTAC/TATA/CTAG-TATT. In the S. typhimurium and E. coli nrdAB genesthis sequence is located between the +1 position and thetranslation start point (Carlson et al., 1984; Tuggle andFuchs, 1986; Jordan et al., 1995), whereas in the nrdEFoperon of both bacteria it overlaps the 710 region(Fig. 1B). The presence of this sequence in the four pro-moters opens up further possibilities and perspectivesfor the analysis of nrdEF expression control.

Experimental procedures

Bacterial strains, phages, and growth conditions

S. typhimurium and E. coli strains and plasmids used in thisstudy are listed in Table 1. The temperature-sensitive mutantof S. typhimurium UA1697, whose mutation had beendescribed to be located in the gyrA–nrdAB region (Maureret al., 1984), was definitively identified as an nrdABts mutantby complementation assays using plasmid pUA447 carryingthe nrdAB operon of S. typhimurium. Cells were grown aero-bically at 378C, unless otherwise stated. The following richand minimal media were used: Luria broth (LB; Roth, 1970),nutrient broth (NB; Miller, 1992), and minimal medium A(MMA; Miller, 1992) supplemented with 0.2% glucose as thecarbon source, and thymidine at 50mg ml71 when necessary.Solid media were prepared by using 1.5% agar. Antibioticswere used at the following concentrations: kanamycin,50mg ml71; tetracycline, 17mg ml71; ampicillin, 50mg ml71;spectinomycin, 100mg ml71; rifampicin, 75mg ml71. Comple-mentation of temperature sensitivity and hydroxyurea hyper-sensitivity were determined as reported elsewhere (Jordanet al., 1994a).

General genetic techniques

Transductions in S. typhimurium were carried out by using ahigh-transduction derivative of phage P22int-4 as describedby Roth (1970). E. coli transductions were performed byusing phage P1vir (Silhavy et al., 1984). Plasmid DNA wastransformed into competent E. coli strains as described by Sil-havy et al. (1984), and into S. typhimurium by electroporationas reported (O’Callaghan and Charbit, 1990).

Construction of chromosomal nrdEF mutants

To construct E. coli nrdEF knockout mutants, an SpcR cas-sette was used. This cassette, obtained from plasmidpHP45OSmR/SpcR (Prentki et al., 1984), was inserted intothe internal Bst XI site of the E. coli nrdE gene carried inpUA446 to give rise to plasmid pUA472. The 5 kb fragmentfrom this plasmid containing the inactivated nrdE gene was




Table 1. Bacterial strains and plasmids.

Strain/PlasmidDescription/Genotype/Constructiona Sourceb/Reference

Strain

E. coli K-12DH5aF ' recA1 endA1 hsdR17

supE44 thi-1 gyrA96relA1 D(lacZYA–argF )U169 deoRf80dlacZM15 F '

Clontech Lab. Inc.

KK450 nrdAts nrdB1 thyA thrleu thi deo tonAlacY supE44 gyrA

B. M. Sjoberg

S17-1 (lpir ) S17-1 lysogenized withl pir bacteriophage

de Lorenzo et al.(1990)

MC1061 DlacX74 hsdR mcrBaraD139 D(araABC–leu )7679 galU galKrpsL thi

M. Casadaban

UA4854 MC1061 Rif R This laboratoryHB101 recA13 hsdS20

supE44 ara-14proA2 lacY1 galK2rpsL20 xyl-5 mtl-1

M. Casadaban

UA4855 HB101 Rif R This laboratoryW3110 F7 l

7 D. DanielsUA6055 UA4854 nrdE ::OSmR/

SpcRThis work

UA6086 KK450 nrdE ::OSmR/SpcR

This work

UA6047 UA4855 mini-Tn5Km-nrdEF (KmR)

This work

UA6069 KK450 mini-Tn5Km-nrdEF (KmR)

This work

UA6087 UA6069 nrdE ::OSmR/SpcR (KmR)

This work

UA6088 UA6069 mini-Tn5Km-nrdE ::OSmR/SpcR

(KmR)

This work

UA6056 UA4855 mini-Tn5Km-nrdE ::lacZ (KmR)

This work

UA6059 MC1061 mini-Tn5Km-nrdE ::lacZ (KmR)

This work(UA6056�MC1061)

S. typhimurium LT2

LT2 Wild type K. SandersonTR2279 recA1 proAB47 pyrB6 J. CasadesusUA1573 TR2279 Rif R This laboratoryDB4708 DB4707 ts-98

(nrdABts) TcRMaurer et al. (1984)

UA1697 LT2 ts-98 (nrdABts)TcR

This work(DB4708� LT2)

MA785 dnaA747ts

zib1257 ::Tn10dtet(TcR)

R. Maurer via L. Bossi

TT1711 thy-283 rec(BC)10zgb18 ::Tn10 (TcR)hisD1447

J. R. Roth via K.Sanderson

CH1701 hns-6 ::Tn10 (TcR) O’Byrne and Dorman(1994)c

CH340 leu-500 ara-9 trp-1016 ::Tn10 (TcR)/pLK1(AmpR)

Richardson et al.(1984)c

CH582 CH340 DtopA2762Trp7 Cys7 Leu+

Richardson et al.(1984)c

CH593 CH340 tos-4 Richardson et al.(1984)c

UA1739 UA1573 mini-Tn5Km-nrdE ::lacZ (KmR)

This work

Table 1. Continued.


UA1740 LT2 mini-Tn5Km-nrdE ::lacZ (KmR)

This work(UA1739�LT2)

UA1741 UA1740 ts-98(nrdABts) TcR

This work(UA1697�UA1740)

UA1742 UA1740 dnaA747ts

zib1257 ::Tn10 dtet(TcR)

This work(RM595�UA1740)

UA1743 UA1740 thy-283zgb18 ::Tn10 (TcR)

This work(TT1711�UA1740)

UA1744 UA1740 hns-6 ::Tn10(TcR)

This work(CH1701�UA1740)

UA1745 CH340 mini-Tn5Km-nrdE ::lacZ (KmR)

This work(UA1740�CH340)





Plasmid

pBluescriptSK(+)

AmpR Stratagene

pGP1-2 Plac ::c I857 PL ::T7RNA polymerase orip15A KmR

Tabor and Richardson(1985)

pHP45OSmR/SpcR

Source of SmR/SpcR

cassette AmpRPrentki and Krisch

(1984)pGP704 ori R6K mob RP4

MCS of M13tg131AmpR

Herrero et al. (1990)

pUC18Not As pUC18 but withMCS flanked by Not Isites AmpR

Herrero et al. (1990)

pUJ8 Promoterless vectorfor making lacZfusions AmpR


pUTmini-Tn5Km Mini-Tn5 Km inplasmid pUT AmpR

KmR


pUA447 pBSK + 4.3 kb (HpaI–Pst I) S.t. nrdABgenes

Jordan et al. (1995)

pUA335 pBSK + 4.9 kb (XmaI–Pst I) S.t. nrdEFgenes

Jordan et al. (1994a)

pUA338 pBSK + 4 kb (by 3'exonuclease IIIdeletion frompUA335) S.t. nrdEFgenes

Jordan et al. (1994a)

pUA426 pBSK + 8 kb (ClaI) E.c.nrdEF genes

This work

pUA425 pBSK + 5 kb (AccI) E.c.nrdEF genes

This work

pUA524 pBSK + 6 kb (EcoRI–ClaI) E.c. nrdEFgenes

This work

pUA523 pBSK + 1.5 kb (EcoRI–AvaI) E.c. nrdEFpromoter region

This work

pUA446 pBSK + 3 kb (AccI–SacII) E.c. nrdEgene

This work

pUA472 pUA446 with OSmR/SpcR inserted inBst XI

This work

pUA474 pGP704 + mutagenized E.c. nrdE gene


then cloned in the pGP704 plasmid and transformed in theS17-1(lpir ) strain. This plasmid is unable to replicate inhost strains devoid of the R6K-specified p protein product ofthe pir gene (Kolter et al., 1978); thus it must be maintainedin bacteriophage lpir lysogenic strains. The recombinant sui-cide plasmid (pUA474) was transferred into Rif R derivativesof E. coli MC1061, UA6069 or KK450 strains. SpcR transcon-jugants were screened for loss of vector-mediated ampicillinresistance to detect putative mutants that had exchangedtheir wild-type gene for the inactivated nrdE as a conse-quence of a double cross-over event (van Haute et al.,1983). Afterwards, the presence of the desired exchangewas unequivocally confirmed by Southern hybridizationexperiments by using a 3 kb DraI–SacII fragment containingthe nrdE gene as a probe.

Insertion in the bacterial chromosome of mini-Tn5transposons carrying either the whole nrdEF operonor nrdE:: lacZ fusions

To obtain an E. coli strain carrying a second chromosomalcopy of the nrdEF genes, the 8 kb insert of plasmid pUA426containing the whole operon was cloned into pUC18Not I,obtaining pUA477. Plasmid pUA479 was then constructedby insertion of the 8 kb Not I fragment containing the nrdEFoperon in the Not I site of suicide plasmid pUTmini-Tn5Km(de Lorenzo et al., 1990). pUTmini-Tn5Km replication de-pends on the pir gene product and can only autonomouslyreplicate in bacteriophage lpir lysogenic strains. The recom-binant plasmid was transferred to a Rif R derivative of E. coli

HB101 (RecA7, to avoid integrative recombination events),and kanamycin-resistant transconjugants were screened forloss of vector-mediated ampicillin resistance to detect clonescontaining an additional copy of the nrdEF operon by transpo-sition. Southern analysis with a probe of the nrdE gene wascarried out with 12 of these clones to confirm that the nativecopy of the operon was intact and that the second nrdEFcopy was in all transconjugants at different locations in thechromosome, keeping one of these clones (UA6047) forfurther studies. Using the same method, the mini-Tn5Km-nrdEF transposon was introduced into strain KK450, givingrise to strain UA6069.

The same procedure was used to insert nrdE ::lacZ fusionsinto the chromosome, but prior to cloning them in pUTmini-Tn5Km, fusions were generated in vector pUJ8 (de Lorenzoet al., 1990). To perform the fusion between the S. typhimur-ium nrdE and the lacZ gene, a 1.7 kb EcoRV fragment con-taining the 5' region of the cloned S. typhimurium nrdEFoperon (Jordan et al., 1994a) was subcloned into plasmidpUJ8. For the E. coli fusion, the 3.5 kb ClaI–AvaI 5'-end frag-ment of the insert contained in pUA426 was subcloned intopUJ8. After insertion of both fusions in plasmid pUTmini-Tn5Km, they were transferred to Rif R derivatives of RecA7

S. typhimurium (TR2279) and E. coli (HB101) strains, respec-tively. The presence of the desired fusion in the chromosomeof both bacteria was confirmed in 12 kanamycin-resistant,ampicillin-sensitive transconjugants of each one by Southernhybridization. Fusions linked to kanamycin resistance weretransduced to RecA+ strains of S. typhimurium (LT2) or E.coli (MC1061) with P22 or P1vir-mediated transduction,respectively.

Beta-galactosidase activity assays

Stationary-phase cultures (grown for 14 h) were subcultured1:100 into fresh media with appropriate antibiotics andgrown with shaking. All assays were started in early logphase-growing cultures (OD600 = 0.05), unless otherwisestated. Samples were taken at the times specified in the indi-vidual experiments, and b-galactosidase activities wereassayed in triplicate as described by Miller (1992). The datapresented in the figures are representative results from atleast two experiments.

DNA manipulations

All restriction enzymes, T4 DNA ligase and polymerase, andthe DIG DNA Labelling and Detection Kit were from Boehrin-ger Mannheim. The conditions used for plasmid and chromo-somal DNA extractions, preparation of lambda phage-liquidlysates, and extraction of DNA from lysates, restriction endo-nuclease digestion, agarose gel electrophoresis, Southernhybridizations, and isolation and ligation of DNA fragmentshave been described elsewhere (Sambrook et al., 1989).

DNA sequencing

Prior to DNA sequencing, a set of exonuclease III-mediatednested deletions of the cloned E. coli insert (pUA523 plasmid)


This workpUA477pUC18Not + 8 kb (ClaI) E.c. nrdEF genesThis workTable 1. Continued.


pUA479 pUTmini-Tn5Km + 8 kb(Not I) E.c. nrdEFgenes

This work

pUA537 pBSK + 1.1 kb (by 5'exonuclease IIIdeletion frompUA523) E.c. nrdEFpromoter region

This work

pUA538 internal 0.1 kb AccIdeletion frompUA537

This work

pUA539 pBSK + 0.9 kb (by 3'exonuclease IIIdeletion frompUA335) S.t. nrdEFpromoter region

This work

pUA451 pUJ8 + 1.7 kb (EcoRV)S.t. nrdEF promoter

This work

pUA454 pUTmini-Tn5Km + S.t.nrdE ::lacZ fusion

This work

pUA461 pUJ8 + 3.5 kb (ClaI–AvaI) E.c. nrdEFpromoter

This work

pUA482 pUTmini-Tn5Km + E.c. nrdE ::lacZ fusion


was created by using the Erase-a-Base system (Promega).The DNA sequence of each of these clones was determinedby the dideoxy method (Sanger et al., 1977) using the DIGTaq sequencing kit and by running the sequencing reactionin a DBE-system GATC 1500 DNA sequencing machine(MWG-Biotech). The entire nucleotide sequence was deter-mined in both DNA strands. Computer analysis was carriedout using the University of Wisconsin Genetics ComputerGroup package (Version 7.2). The sequence of the 1529 bpfragment belonging to the E. coli nrdEF operon describedhere has been submitted to the EMBL/GenBank/DDBJNucleotide Sequence Data Libraries under Accession Num-ber X79787.

RNA extraction

Total RNA for Northern and primer extension analysis wasprepared by the hot acidic phenol method (Wong and Clel-land, 1994) from E. coli and S. typhimurium strains grownexponentially in rich medium to an OD600 of 0.5, and treatedwith RNase-free DNase I (Boehringer Mannheim) prior touse. RNA concentration and integrity were determined byA260 measurements and 1% formaldehyde–agarose gel elec-trophoresis, respectively (Sambrook et al., 1989).

RNA analysis by Northern hybridization

DIG-labelled S. typhimurium and E. coli nrdEF mRNA specificriboprobes were made by linearizing pUA338 with Pst I andpUA446 with EcoRI, respectively, and transcribing the linear-ized template with the T3 RNA polymerase using a DIG RNA-labelling mixture (Boehringer Manheim). Expression levelsand sizes of nrdEF mRNA were determined by Northernhybridization analysis after transfer of formaldehyde–agar-ose electrophoresed RNA to nylon membranes (BoehringerManheim) using alkaline transfer as described by Low andRausch (1994). Prehybridization, hybridization with the DIG-labelled nrdEF-specific riboprobe at 688C, stringencywashes, and probe signal detection with the AMPPD chemilu-minescence system (Boehringer Manheim) were carried outas specified by the suppliers.

Primer extension analysis of mRNA

For S. typhimurium, oligonucleotides 5'-ATCGCGTTCAGG-GCGTGGTAATCC-3' (St1), 5'-CGGTGCGTATTTTCAGA-3'(St2), 5'-AAGCCAGACCAGCTCAAATC-3' (St3) and 5'-GCTCATGATTCGTATTTCC-3' (St4), which hybridize fromnucleotides + 42 to + 65, 7 353 to 7 337, 7454 to 7435and 7638 to 7620 relative to the nrdE translational startpoint, respectively, were used. For E. coli, the oligonucleotideused was 5'-GCGCATGATTCGTATTTCC-3' (Ec1), whichhybridizes from nucleotide 7638 to 7620 (Fig. 1B). All pri-mers were synthesized and 5'-end labelled with DIG-NHS-Ester (MWG-Biotech).

For S. typhimurium, and after using several oligonucleo-tides cited above, we obtained the same primer extension sig-nal with primers St3 and St4 . For E. coli we used primer Ec1,which is analogous to St4, and we obtained the same signalas for S. typhimurium.

Primer extension was carried out as follows: 50mg of total

RNA was hybridized with 4 pmol 5'-end DIG-labelled primerin a 15ml final volume containing 3ml of 5� AMV-RT buffer(Promega) by denaturing it at 908C for 1 min, followed by3 min at 678C, cooling it down to 458C slowly, and maintainingit for a further 10 min at this temperature. Afterwards, 15ml ofextension solution was added, containing 3ml of 5� AMV-RTbuffer, 20 U of AMV reverse transcriptase (Promega), 40 U ofRNase inhibitor (Boehringer), and 0.66 mM each of dATP,dCTP, dGTP and dTTP. The reaction was incubated at458C for 90 min, after which it was stopped with 1ml of 0.5 MEDTA (pH 8). Free RNA was removed by treatment withRNase for 15 min at 378C and the DNA was ethanol-precipitated and redissolved in 6ml of formamide loadingdye. A total of 2ml of the sample was loaded and separatedon a 6% denaturing polyacrylamide gel by using the DBE-system GATC 1500 DNA sequencing machine (MWG-Biotech). A DNA sequencing ladder, obtained using thesame primer, was electrophoresed alongside the productsof the primer extension assay for calibration.

Semi-quantitative competitive RT-PCR for titration ofnrdEF transcription

For the quantification of nrdEF expression in E. coli cells bycompetitive PCR, the following synthesized oligonucleotideswere used as primers. Oligonucleotide 5'-CAGCGCGT-GGTAATCCAT-3' (RTlow), complementary to nucleotides+ 40 to + 57 relative to the nrdE translation start site, wasemployed for initial primer extension. Oligonucleotide 5'-GCGATCTTAGCTGGTCTG-3' (RTup) was identical to nuc-leotides 7456 to 7439. PCR amplification of the cDNA pro-duct with both primers generated a 513 bp fragment.

This technique uses co-amplification of a known amount ofa homologous DNA fragment to normalize differences inamplification efficiencies among samples. A 414 bp DNA stan-dard for competitive PCR was obtained by amplification, withthe same set of primers, of plasmid pUA538, constructed byinternal deletion of a 99 bp AccI fragment from plasmidpUA537. After isolation by electrophoresis and spectrophoto-metric quantification at A260, several dilutions of the fragmentwere used as the DNA competitor.

A simple miniprep method for obtaining total RNA fromexponentially grown E. coli cells described by Garrido et al.(1993) was used to obtain DNA-free RNA. Total RNA wasextracted from 5�105 to 5�106 cells and finally resus-pended in 50ml of deionized water. RNA samples werechecked for DNA contamination by the same proceduredescribed below, without the addition of reverse transcriptase.Competitive RT-PCR was carried out as described by Gilli-land et al. (1990), with minor modifications. For initial primerextension, 5ml aliquots of total RNA samples were mixedwith 2ml of 10� PCR buffer (Boehringer Manheim), 4ml ofdNTP mix (2 mM each), and 25 pmol primer RTlow in a finalvolume of 20ml. After incubation at 658C for 5 min, primerannealing was performed at room temperature for 5 min,and RNase inhibitor (4 U) and AMV reverse transcriptase(10 U) were added to perform cDNA synthesis at 428C for60 min. Finally, the reaction was stopped by heating at 958Cfor 5 min and chilling on ice. All incubations were carried outusing a programmed DNA Thermal Cycler 480 (Perkin-Elmer).



Amplification was performed by adding 2ml of the cDNAsamples to 50ml of 1� PCR buffer (Boehringer Manheim)containing 0.2 mM of each dNTP, 0.5mM of each of theRTup and RTlow primers, 1.5 U of Taq DNA polymeraseand a constant amount of the competitor. After incubationfor 3 min at 948C, samples were subjected to 30 cycles ofamplification (1 min at 948C, 1 min at 558C and 1 min at728C), followed by 7 min at 728C. Reaction products (15ml)were resolved in ethidium bromide–3% NuSieve/agarosegels run at 5 V cm71, photographed, and analysed by densito-metry. The levels of each product were corrected for its size tocompensate label incorporation. As indicated by Garrido et al.(1993), the presence of hybrid molecules constituted by com-plementary strands of the competitor and wild-type moleculesis of minor importance when relative quantification is required.Afterwards, the ratio of amplified target to competitor PCRproducts was determined for each sample. Variations in theratios indicate the relative differences in initial mRNA levelsbetween the samples, as previously reported by Price et al.(1992).

Expression of plasmid-encoded proteins

Expression and selective [35S]-methionine labelling of plas-mid-encoded polypeptides in a bacteriophage T7 RNA poly-merase expression system were carried out as describedby Tabor and Richardson (1985). Cell lysates were sepa-rated on a 16.5% SDS–polyacrylamide gel run with a Tris–Tricine–SDS buffer as described by Scagger and vonJagow (1987) for superior resolution of small proteins at100 V on a modular Mini-Protean II electrophoresis system(Bio-Rad). The gel was dried and autoradiographed.

Acknowledgements

This work was funded by grants PB91-0470 and PB94-0687of the Direccion General de Investigacion Cientıfica y Tecnicaof Spain (DGICYT) and partially supported by the Comissio-nat per Universitats i Recerca de la Generalitat de Catalunya.A.J. was a recipient of a predoctoral fellowship from theDireccio General d’Universitats de la Generalitat de Cata-lunya. We gratefully acknowledge the help of the DireccioGeneral d’Universitats de la Generalitat de Catalunya for dif-ferent grants for the purchase of equipment. We are indebtedto Dr Peter Reichard for his suggestions and critical reading ofthe manuscript.

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Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria