Expression Analysis of the nrdHIEF Operon fromEscherichia coli

Abstract

Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB andnrdHIEF operons. While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function. Here, we present evidence that nrdHIEF expression might be important under certain circumstances. Basal transcript levels were dramatically enhanced (25–75-fold), depending on the growth-phase and the growth-medium composition. Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants. Moreover, nrdHIEFexpression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium). The mechanism(s) that triggers nrdHIEFexpression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of thenrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. colimight optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors. Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB andnrdHIEF operons. While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function. Here, we present evidence that nrdHIEF expression might be important under certain circumstances. Basal transcript levels were dramatically enhanced (25–75-fold), depending on the growth-phase and the growth-medium composition. Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants. Moreover, nrdHIEFexpression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium). The mechanism(s) that triggers nrdHIEFexpression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of thenrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. colimight optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors. ribonucleotide reductase thioredoxin glutaredoxin polymerase chain reaction reverse transcription-PCR paraquat t-butyl hydroperoxide 4-nitroquinoline 1-oxide Ribonucleotide reductases (RRases)1 provide the building blocks for DNA biosynthesis in all living organisms. RRases are grouped into three major classes (class I, II, and III) based on the mechanisms they use for radical generation and on their structural differences. Class I is subdivided further into two subclasses (Ia and Ib) based mainly on allosteric regulation but also on involvement of auxiliary proteins (recently reviewed in Ref. 1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (626) Google Scholar). Escherichia coli has the coding potential for three different RRases. The NrdAB (class Ia) is active during aerobiosis, NrdDG (class III) is strictly anaerobic, and NrdEF (class Ib) is thought to be a cryptic enzyme with no obvious function (1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (626) Google Scholar). Class I enzymes receive the electrons required for the reduction of ribose from small proteins, thioredoxins (Trxs) and glutaredoxins (Grxs), with two redox-active cysteine thiols, which by dithiol-disulfide interchange reduce an acceptor disulfide in the active center of RRase. Trxs and Grxs are kept in a reduced form by NADPH, which reduces the redoxin either via the flavoprotein thioredoxin reductase or via the flavoprotein glutathione reductase and the ubiquitous tripeptide GSH (2Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). Escherichia colicontains two thioredoxins (Trx1 and Trx2), three glutaredoxins (Grx1, Grx2, and Grx3), and a novel redoxin (called NrdH) with thioredoxin-like activity but glutaredoxin-like amino acid sequence (2Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 3Miranda-Vizuete A. Damdimopoulos A.E. Gustafsson J.-A. Spyrou G. J. Biol. Chem. 1997; 272: 30841-30847Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 4Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar, 5Jordan A. Åslund F. Pontis E. Reichard P. Holmgren A. J. Biol. Chem. 1997; 272: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Grx1 and Trx1 are the two main hydrogen donors of the E. coli NrdAB enzyme (3Miranda-Vizuete A. Damdimopoulos A.E. Gustafsson J.-A. Spyrou G. J. Biol. Chem. 1997; 272: 30841-30847Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 4Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar, 6Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 7Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar). NrdH is a more specific hydrogen donor for the NrdEF than for the NrdAB enzyme, whereas the opposite is the case for Grx1 (5Jordan A. Åslund F. Pontis E. Reichard P. Holmgren A. J. Biol. Chem. 1997; 272: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Trx1 and Trx2 are hydrogen donors for NrdAB but not for NrdEF (3Miranda-Vizuete A. Damdimopoulos A.E. Gustafsson J.-A. Spyrou G. J. Biol. Chem. 1997; 272: 30841-30847Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 5Jordan A. Åslund F. Pontis E. Reichard P. Holmgren A. J. Biol. Chem. 1997; 272: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 8Jordan A. Pontis E. Atta M. Krook M. Gibert I. Barbé J. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12892-12896Crossref PubMed Scopus (98) Google Scholar). The nrdA and nrdB genes that code for the NrdAB class Ia reductase constitute a tightly regulated transcription unit that does not include the gene for either Trx or Grx. Expression ofnrdAB genes is cell cycle-regulated and increases when DNA synthesis is inhibited (1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (626) Google Scholar). Regulation of nrdAB expression in E. coli has been shown to be very complex; multiplecis-acting positive regulatory sites identified upstream of the nrdAB promoter appear to control, independently but in concert, the cell cycle-dependent transcription and the response to inhibition of DNA synthesis (9Jacobson B.A. Fuchs J.A. Mol. Microbiol. 1998; 28: 1315-1322Crossref PubMed Scopus (23) Google Scholar). Recent data from our group add further complexity to the nrdAB regulation, demonstrating a tight and inverse relation between expression ofnrdAB and that of genes coding for components of both glutaredoxin and thioredoxin pathways (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Interestingly, we have observed that induction ratios of nrdAB transcription by hydroxyurea (RRase inhibitor) are similar to the increments innrdAB basal expression of mutants lacking both Trx1 and Grx1. Therefore, we have postulated that operation of the NrdAB enzyme in the absence of its two main reductants must lead to disturbances in deoxyribonucleotide production sensed as those caused by hydroxyurea. The nrdE and nrdF genes that code for the NrdEF class Ib reductase form a conserved operon where the promoter is followed by four genes (11Jordan A. Aragall E. Gibert I. Barbé J. Mol. Microbiol. 1996; 19: 777-790Crossref PubMed Scopus (68) Google Scholar). Genes nrdH, coding for the NrdH-redoxin, and nrdI, coding for a protein with a stimulatory effect on ribonucleotide reduction, are present upstream ofnrdEF (5Jordan A. Åslund F. Pontis E. Reichard P. Holmgren A. J. Biol. Chem. 1997; 272: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). It has been reported that the E. colitranscription unit is not expressed in sufficient amounts to support growth under normal laboratory conditions. Neither inhibitors of DNA replication nor DNA damages induce its expression. Thus far, only hydroxyurea has been shown to stimulate the expression ofnrdEF genes (11Jordan A. Aragall E. Gibert I. Barbé J. Mol. Microbiol. 1996; 19: 777-790Crossref PubMed Scopus (68) Google Scholar). We have quantitated the in vivo transcription of genes encoding for components of both glutaredoxin and thioredoxin pathways by means of a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) approach (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 12Gallardo-Madueño R. Leal J.F.M. Dorado G. Holmgren A. López-Barea J. Pueyo C. J. Biol. Chem. 1998; 273: 18382-18388Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In this protocol, all target genes, a housekeeping gene (also named control gene, reference gene, or internal standard), and one external standard are amplified in the same reaction tube. Specific fluorescent primers are used, and amplification products are analyzed with a DNA sequencer. Putative variations in the expression of the housekeeping gene are controlled by the external standard. Relative expressions of the targets to the reference (or to the external standard) are measured. Lately (13Pueyo C. Jurado J. Prieto-Álamo M.J. Monje-Casas F. López-Barea J. Methods Enzymol. 2001; Google Scholar), we have experimentally demonstrated that our methodology fulfills all theoretical requirements for precise quantification of both induction and repression of gene transcription. Because of the PCR amplification step, our method displays a much higher sensitivity than those of current techniques for mRNA quantitation, like Northern blotting or primer extension analyses. Here, we used this sensitive experimental approach to identify those growth conditions and stress circumstances under which expression of nrdHIEF genes might be required inE. coli. All bacterial strains were E. coli K-12. UC5710 (arg56, nad113, araD81, Δ(uvrB-bio)) was considered the parental wild type (14Abril N. Roldán-Arjona T. Prieto-Álamo M.J. van Zeeland A.A. Pueyo C. Environ. Mol. Mutagen. 1992; 19: 288-296Crossref PubMed Scopus (29) Google Scholar). UC1342 (ΔoxyR::kan), UC1394 (oxyR2), UC1333 (Δsox-8::cat), UC827 (ΔtrxA grxA::kan), UC1358 (ΔtrxA gshA), UC1363 (ΔtrxA gshAΔoxyR::kan), UC1395 (ΔtrxA gshA oxyR2), UC1101 (pKM101), UC498 (katG katE::Tn10 pKM101), and UC628 (((sodA::Mud PR13)25 (sodB-kan)1-Δ2) pKM101) have been previously described (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 15Miranda-Vizuete A. Rodriguez-Ariza A. Toribio F. Holmgren A. López-Barea J. Pueyo C. J. Biol. Chem. 1996; 271: 19099-19103Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 16Prieto-Álamo M.J. Abril N. Pueyo C. Carcinogenesis. 1993; 14: 237-244Crossref PubMed Scopus (29) Google Scholar). UC499 (katG17::Tn10), UC4910 (ΔahpCF::kan), UC1049 (katG katE::Tn10), and UC4101 (katG17::Tn10ΔahpCF::kan) were constructed by P1-mediated transduction. Successful transfer of thekatG17::Tn10 mutation was confirmed by assaying catalase hydroperoxidase I activity, as described (17Meir E. Yagil E. Curr. Microbiol. 1985; 12: 315-320Crossref Scopus (9) Google Scholar). Successful transfer of the ΔahpCF::kan deletion was confirmed by screening for no DNA amplification with specific primers (18Michán C. Manchado M. Dorado G. Pueyo C. J. Bacteriol. 1999; 181: 2759-2764Crossref PubMed Google Scholar). Strains with the ΔoxyR::kan or Δsox-8::cat mutant allele do not induce the expression of genes regulated by OxyR or SoxR/S upon exposure to H2O2 or paraquat, respectively (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Strains carrying the oxyR2 allele exhibit constitutive high levels of OxyR-regulated gene expression (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Strains with the ΔtrxA and with either thegrxA::kan or the gshA mutant allele exhibit undetectable levels of Trx1 and of Grx1 or GSH, respectively (15Miranda-Vizuete A. Rodriguez-Ariza A. Toribio F. Holmgren A. López-Barea J. Pueyo C. J. Biol. Chem. 1996; 271: 19099-19103Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Double katG katE or sodA sodBmutants retain <1% of the wild-type catalase or superoxide dismutase level, respectively (16Prieto-Álamo M.J. Abril N. Pueyo C. Carcinogenesis. 1993; 14: 237-244Crossref PubMed Scopus (29) Google Scholar). Double katG ahpCF mutants are known to have increased intracellular levels of peroxides (19Rosner J.L. Storz G. Antimicrob. Agents Chemother. 1994; 38: 1829-1833Crossref PubMed Scopus (37) Google Scholar, 20Åslund F. Zheng M. Beckwith J. Storz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6161-6165Crossref PubMed Scopus (437) Google Scholar). Plasmid pKM101 carries the mucAB genes, which make bacteria more susceptible to SOS-dependent mutagenesis (21Ruiz-Laguna J. Prieto-Álamo M.J. Pueyo C. Environ. Mol. Mutagen. 2000; 35: 22-30Crossref PubMed Scopus (17) Google Scholar). Bacteria were grown in Luria-Bertani (LB) nutrient broth or M9 minimal medium (glucose at 2 g/liter), as described (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). E. coli cells from an overnight culture in LB broth were centrifuged and diluted 100-fold into 50 ml of M9 minimal medium and incubated at 37 °C and 150 rpm to reach anA600 of 0.2. At this stage, the bacteria were further grown in the absence or presence of hydroxyurea, hydrogen peroxide (H2O2), paraquat (PQ),t-butylhydroperoxide (tBOOH), 4-nitroquinoline 1-oxide (4NQO), orN-methyl-N′-nitro-N-nitrosoguanidine for a fixed time period. The cells were then rapidly cooled to 0 °C for total RNA purification. Primers (listed in Table I) were designed with the Oligo 6.1.1/98 (Molecular Biology Insights Inc., Plymouth, MN) program in order to obtain the highest specificity and performance in multiplexed PCRs. Target genes code for the NrdH-redoxin (nrdH), the NrdI stimulatory protein (nrdI), and the R1E (nrdE) and R2F (nrdF) subunits ofE. coli class Ib RRase. As described previously (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 12Gallardo-Madueño R. Leal J.F.M. Dorado G. Holmgren A. López-Barea J. Pueyo C. J. Biol. Chem. 1998; 273: 18382-18388Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), the gapA gene, which codes ford-glyceraldehyde-3-phosphate dehydrogenase, was used as an internal standard. The internal standard normalizes variations in RNA extraction, reverse transcription, and PCR amplification among samples. To evaluate the extent of putative variations in the expression of the reference gene, an external standard was included, as described (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The external standard was an in vitro synthesized RNA fragment encoded by the CYP1A gene from Liza aurata. This external standard has no homology with the E. coli genome.Table IPCR primer characteristicsPrimerFragment sizeSequencebpExternal standard*935′-TCCTTCAACCCAGACCGTTTCCTCA-3′External standard5′-CCGCTTTCCCAAGCCAAAAACCATC-3′nrdH*1055′-GTTGCGTGCTCAGGGCTTTCGTCAGT-3′nrdH5′-TGTGGCGCTGGATGCAGACGGTTAAT-3′nrdI*1125′-GCCAGCTCGTCTACTTCTCCAGCAGC-3′nrdI5′-TCTACCTGAATCCGTTCCCGCTCATT-3′nrdE*1305′-CGTCCGAACAGTGTGACCTTCAGTAGCC-3′nrdE5′-GTGCGTGGGTAAACAGCGTAATGACAAA-3′nrdF*1365′-GGCTACGAACCGTTATTTCCCGCAGAA-3′nrdF5′-CAACCGCTTTCCCCATCACATAAGAGG-3′gapA*1435′-CGTTCTGGGCTACACCGAAGATGACG-3′gapA5′-AACCGGTTTCGTTGTCGTACCAGGA-3′Forward primers (marked with an asterisk) were labeled with 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein. The housekeepinggapA gene was used as internal standard. Open table in a new tab Forward primers (marked with an asterisk) were labeled with 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein. The housekeepinggapA gene was used as internal standard. RNA purification and cDNA synthesis were as described (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). At least two independent RNA preparations were isolated for each experimental condition, each RNA sample being retrotranscribed on three separated occasions. PCR amplification of cDNA was carried out using all of the primer pairs listed in Table I or using just those for the nrdH and nrdE target genes, thegapA reference gene, and the external standard. In this last case, the multiplex PCR amplification was performed in a mixture (25-μl final volume) containing MPCR buffer 3 (Maxim Biotech Inc., San Francisco, CA) supplemented with 1 mmMgCl2, a 250 μm concentration of each dNTP, 1 μl of cDNA solution, 1.25 units of AmpliTaq Gold DNA polymerase, and primers at the following concentrations: 0.07 μm(nrdH), 0.09 μm (nrdE), 0.04 μm (gapA), and 0.14 μm (external standard). After incubation at 95 °C for 12 min (for activation of DNA polymerase), 26 cycles of PCR were performed. Each cycle consisted of 1 min of denaturation at 94 °C and 45 s of annealing and enzymatic primer extension at 70 °C. These multiplex PCR conditions were optimized as detailed (13Pueyo C. Jurado J. Prieto-Álamo M.J. Monje-Casas F. López-Barea J. Methods Enzymol. 2001; Google Scholar) to ensure that the amplifications were in the exponential phase and that the efficiencies remained constant in the course of the PCR. Reaction products were quantified as described previously (12Gallardo-Madueño R. Leal J.F.M. Dorado G. Holmgren A. López-Barea J. Pueyo C. J. Biol. Chem. 1998; 273: 18382-18388Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Differences among PCR outcomes were normalized by comparing the fluorescence intensity of each band to that resulting from gapA amplification (internal standard). The levels ofgapA in reference to the external standard remained essentially equal among the strains and experimental conditions investigated in this work. Consequently, changes detected with reference to the housekeeping gapA gene were accurately attributed to variations in the expression levels of the target genes under analysis. Samples for comparison of different experimental conditions or different bacterial strains were handled in parallel. Data are the means ± S.E. from n ≥ 6 independent multiplexed PCR amplifications. Statistical comparisons were done by a hierarchical analysis of variance with SAS software (Statistical Analysis System, version 6.03). Data presented here as relative expression ratios do not provide any indication of the mechanism that contributes to the dynamic control of a particular mRNA concentration, whether it is the rate of transcription initiation or the rate of transcript turnover. In a prior study (11Jordan A. Aragall E. Gibert I. Barbé J. Mol. Microbiol. 1996; 19: 777-790Crossref PubMed Scopus (68) Google Scholar), expression of nrdEF genes was quantified by a competitive RT-PCR, in which the competitor had an internal deletion of 99 base pairs and the reaction products were resolved in ethidium bromide-agarose gels and analyzed by densitometry. Several points at which errors can occur by using such a methodology include differences in amplification efficiencies because of the great size difference between the competitor and the target, possible heteroduplex formation, and the need to compensate for differences in fragment label incorporation. When measuring rare species of mRNA, which requires a high number of PCR cycles, the control of those problems is of maximal importance, even for relative quantification (e.g.see Refs. 22Freeman W.M. Walker S.J. Vrana K.E. BioTechniques. 1999; 26: 112-125Crossref PubMed Scopus (857) Google Scholar, 23Raeymaekers L. Anal. Biochem. 1993; 214: 582-585Crossref PubMed Scopus (215) Google Scholar, 24McCulloch R.K. PCR Methods PubMed Scopus Google Scholar). In this by of the high and of our multiplex (13Pueyo C. Jurado J. Prieto-Álamo M.J. Monje-Casas F. López-Barea J. Methods Enzymol. 2001; Google Scholar), we variations in basal levels expression and in response to stress conditions. To this we confirmed that transcription from the nrdHIEF promoter in wild-type cells and increases upon hydroxyurea exposure (11Jordan A. Aragall E. Gibert I. Barbé J. Mol. Microbiol. 1996; 19: 777-790Crossref PubMed Scopus (68) Google Scholar). UC5710 cells to of hydroxyurea from 1 to for the expression of all of the genes that constitute operon was by using the primer in Table I. Basal levels of and nrdF gene expression were detected and increased upon hydroxyurea not As an an induction level of ± to was observed at the minimal of 1 It is that in the prior study by (11Jordan A. Aragall E. Gibert I. Barbé J. Mol. Microbiol. 1996; 19: 777-790Crossref PubMed Scopus (68) Google Scholar), such an induction level was observed under much more conditions grown in the presence of 50 we do not differences in the number of that expression of gene was increased by hydroxyurea, the multiplex PCR was in further by just two and of the four genes in transcription unit to the internal and external The expression of nrdH genes the growth of wild-type UC5710 cells in LB medium are shown in expression levels were observed at the of exponential growth then gene expression levels as the culture from to phase ≥ and (also named or are two regulatory proteins with growth expression in LB medium R. J. Bacteriol. 1992; PubMed Google M. A. J. Bacteriol. PubMed Scopus Google Scholar). To the of and on and nrdE transcript levels during the course of cell growth in both mutants were analyzed in comparison with wild-type Data that the presence of nor the presence of the expression of and genes in E. coli. variations in gene expression were observed also with to the of the growth medium bacteria on M9 minimal medium with had from to higher levels of nrdE than bacteria on LB medium control intracellular levels J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), we the on nrdH of the of to the LB nutrient medium 2 g/liter), and of of by as the in the M9 minimal on was observed not As variations were for and nrdE nrdH data be considered of the transcription In to the function of as reductants for both the Trx and pathways are required to the potential of the bacterial F. Zheng M. Beckwith J. Storz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6161-6165Crossref PubMed Scopus (437) Google Scholar, Åslund F. Holmgren A. Beckwith J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the hydroperoxide and superoxide dismutase activities the of peroxides and superoxide their G. J.A. Curr. Microbiol. 1999; PubMed Scopus Google Scholar). The of missing components of on basal levels of nrdHIEFexpression is in Table increments in the levels of were detected in mutants either in both the and pathways or in the of particular is the large quantified in bacteria lacking Trx1 in with either Grx1 or GSH or missing both catalase hydroperoxidase I and hydroperoxide reductase activities levels of gene expression in bacteria in Trx and pathways or in ± ± ± ± in Trx and ± ± ± ± in ± ± ± ± ± ± were grown in LB broth to reach an of Data are the ± S.E. of the fluorescent of target sequence relative to that of standard). type indicates increments relative to wild type Open table in a new tab were grown in LB broth to reach an of Data are the ± S.E. of the fluorescent of target sequence relative to that of standard). type indicates increments relative to wild type OxyR can be either by with an such as H2O2 or by a in the caused by the of the two and pathways F. Zheng M. Beckwith J. Storz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6161-6165Crossref PubMed Scopus (437) Google Scholar, Åslund F. Holmgren A. Beckwith J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), it possible that the induction of nrdHIEF that we in was to activation of This was investigated by the of mutant on basal levels of gene expression in As shown in Table II, expression genes was by in regulatory but it followed a opposite to that by OxyR-regulated genes (10Prieto-Álamo M.J. Jurado J. Gallardo-Madueño R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, G. J.A. Curr. Microbiol. 1999; PubMed Scopus Google Scholar). Therefore, the high basal level of nrdHIEF in UC1358 was further in its ΔoxyR::kan mutant where a increase in the transcript was the a