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Journal of Bacteriology, February 2009, p. 985-995, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01409-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Function and Regulation of Class I Ribonucleotide Reductase-Encoding Genes in Mycobacteria ^,

Mohube B. Mowa,¹ Digby F. Warner,¹ Gilla Kaplan,² Bavesh D. Kana,^1* and Valerie Mizrahi^1*

MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, School of Pathology, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg 2000, South Africa,¹ Laboratory of Mycobacterial Immunity and Pathogenesis, Public Health Research Institute, International Center for Public Health, 225 Warren Street, Newark, New Jersey 07103-3535²

Received 8 October 2008/ Accepted 6 November 2008

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Ribonucleotide reductases (RNRs) are crucial to all living cells, since they provide deoxyribonucleotides (dNTPs) for DNA synthesis and repair. In Mycobacterium tuberculosis, a class Ib RNR comprising nrdE- and nrdF2-encoded subunits is essential for growth in vitro. Interestingly, the genome of this obligate human pathogen also contains the nrdF1 (Rv1981c) and nrdB (Rv0233) genes, encoding an alternate class Ib RNR small (R2) subunit and a putative class Ic RNR R2 subunit, respectively. However, the role(s) of these subunits in dNTP provision during M. tuberculosis pathogenesis is unknown. In this study, we demonstrate that nrdF1 and nrdB are dispensable for the growth and survival of M. tuberculosis after exposure to various stresses in vitro and, further, that neither gene is required for growth and survival in mice. These observations argue against a specialist role for the alternate R2 subunits under the conditions tested. Through the construction of nrdR-deficient mutants of M. tuberculosis and Mycobacterium smegmatis, we establish that the genes encoding the essential class Ib RNR subunits are specifically regulated by an NrdR-type repressor. Moreover, a strain of M. smegmatis mc²155 lacking the 56-kb chromosomal region, which includes duplicates of nrdHIE and nrdF2, and a mutant retaining only one copy of nrdF2 are shown to be hypersensitive to the class I RNR inhibitor hydroxyurea as a result of depleted levels of the target. Together, our observations identify a potential vulnerability in dNTP provision in mycobacteria and thereby offer a compelling rationale for pursuing the class Ib RNR as a target for drug discovery.

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Despite the availability of an effective chemotherapeutic regimen, tuberculosis remains one of the leading causes of death worldwide (15, 19). The massive burden of this disease, combined with the emergence and spread of strains of Mycobacterium tuberculosis that are resistant to first- and second-line drugs (22), underscores the urgent need to develop new treatment-shortening antitubercular drugs with novel modes of action (70). This need is driving efforts to identify, validate, and prioritize new targets for tuberculosis drug discovery (1, 39, 67).

Ribonucleotide reductases (RNRs) play an essential role in all living cells by catalyzing the reduction of ribonucleoside-5'-di- or triphosphates to generate the deoxyribonucleotides (dNTPs) required for DNA replication and repair (44). Given their central role in cellular metabolism, RNRs have attracted considerable interest as targets for novel antiviral, antibacterial, and antiproliferative chemotherapeutics (45, 52, 59, 61, 68). Three distinct classes (I, II, and III) of RNRs have been defined; although they conserve the same basic catalytic mechanism, these classes are distinguished by their subunit composition, as well as by the cofactor and oxygen requirements for generating the transient thiyl radical required to activate the ribonucleotide by abstracting the 3' hydrogen of the ribose (36, 44). Class I RNRs are further divided into class Ia, class Ib, and class Ic enzymes (27), comprising separate catalytic (the large, or R1, subunit; NrdA in classes Ia and Ic or NrdE in class Ib) and radical-generating (the small, or R2, subunit; NrdB or NrdF) subunits. The class Ic RNR was recently identified in Chlamydia trachomatis (27) and is defined by its replacement of the catalytically essential tyrosyl radical residue of the classical class I R2 subunit with a phenylalanine and its use of a stable Fe(IV)-Fe(III) or Mn(IV)-Fe(III) cofactor to directly initiate production of the cysteinyl radical in the R1 subunit (31, 62). It has been hypothesized that the use of this alternate cofactor might render the enzyme more resistant to reactive nitrogen and oxygen species (27), including the antimicrobial effector nitric oxide (NO), which targets the tyrosyl radical (21). Recent studies of the class Ic RNR from Chlamydia trachomatis support this notion (31), suggesting that this form of the enzyme might enable intracellular pathogens to survive in the face of the nitrosative and oxidative stresses imposed by the host immune response (27).

Many organisms possess more than one class of RNR (32-34), suggesting that different enzymes might function to allow adaptation to varying oxygen levels in the environment (47, 49). However, different RNR classes have been shown to be active simultaneously in some organisms during aerobic growth (4, 5, 34), and a number of bacterial genomes contain more than one enzyme of the same class or subclass (33, 37, 40). The complement of RNR-encoding genes in sequenced mycobacteria reveals both common and unique features (Fig. 1) (http://rnrdb.molbio.su.se). All possess a class Ib RNR encoded by nrdE and a genetically linked nrdF gene, designated nrdF2 (Rv3048c in the reference organism, M. tuberculosis H37Rv [14]). nrdE is the only R1-encoding gene identified in mycobacteria. Species other than Mycobacterium leprae and Mycobacterium ulcerans also possess an R2 subunit-encoding gene homologous to that of the chlamydial class Ic RNR (27), designated nrdB (Rv0233 in M. tuberculosis H37Rv [14]). M. tuberculosis and Mycobacterium bovis are distinguished by the presence of both an alternate class Ib R2 subunit-encoding gene, nrdF1 (Rv1981c in H37Rv), and a class II RNR-encoding gene, nrdZ (Rv0570 in H37Rv) (14, 16) (Fig. 1). Mycobacterium smegmatis mc²155, on the other hand, carries nrdH, nrdI, nrdE, and nrdF2 on a 56-kb duplicated region of the genome, which endows this organism with two copies of each of these genes (64).

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FIG. 1. Composition and organization of RNR-encoding genes in M. tuberculosis H37Rv and M. smegmatis mc2155. The genes are denoted by shaded or filled arrows. NrdR boxes upstream of the nrdH and nrdF2 genes are shown as hatched boxes. The thick vertical bars in the M. smegmatis diagram represent the IS1096 elements flanking the 56-kb chromosomal duplication that harbors the class Ib RNR-encoding genes (64). The gene names and open reading frame (Rv or MSMEG) numbers are taken from TubercuList (http://genolist.pasteur.fr/TubercuList/) and GenBank accession number NC_008596 and are shown above and below the genes, respectively. The intergenic spacing between the class Ib RNR-encoding gene clusters is shown. Unlike that in M. tuberculosis, the nrdI gene in M. smegmatis (MSMEG_1018 and MSMEG_2298) is a pseudogene.

Given the multiplicity of RNR-encoding genes in M. tuberculosis and their transcriptional responsiveness to different stresses (6, 48, 63), we hypothesized that this organism may be able to fine-tune the provision of dNTPs for DNA replication and repair to the varying environmental conditions that it encounters (Fig. 2). In prior work, we showed that nrdF2 is essential for aerobic growth of M. tuberculosis H37Rv in vitro, confirming that the class Ib NrdEF2 enzyme provides the principal RNR function under these conditions (16). We also showed that deletion of nrdZ had no effect on the growth or survival of M. tuberculosis under the conditions of hypoxia in which the gene is induced or on the virulence of the organism in mice (16). In the present study, we adopted a genetic approach to investigate whether nrdB and nrdF1 may play specialist roles in dNTP supply in M. tuberculosis. Our findings further validate the NrdEF2 enzyme as a novel target for antitubercular drug discovery (16, 45) and demonstrate a role for the NrdR protein (5, 60) as a specific repressor of the essential class Ib RNR-encoding genes. We also show that a mutant of M. smegmatis mc²155 that lacks the 56-kb chromosomal duplication (64) and another mutant, containing only one functional copy of nrdF2, are hypersensitive to the class I RNR inhibitor hydroxyurea (HU). This phenotype is shown to be directly attributable to the reduced dosage of genes that encode the NrdEF2 enzyme, reinforcing the apparently dominant role of the class Ib enzyme in nucleotide cycling and dNTP pool modulation in mycobacteria.

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FIG. 2. Role of putative alternate RNRs in the provision of dNTPs in M. tuberculosis. The class Ib NrdEF2 enzyme is essential for aerobic growth of M. tuberculosis in vitro (16). The biochemical characteristics of the chlamydial class Ic RNR (30) and the predicted lifestyle of M. tuberculosis suggest that NrdB may serve a specialist function in dNTP supply under conditions of nitrosative stress. The class II RNR-encoding gene nrdZ belongs to the regulon of "dormancy" genes controlled by the DosRST two-component regulator system and induced by hypoxia and low-dose NO (51, 63) but is dispensable for growth and survival in mice (16). Drug-mediated inhibition of translation or DNA gyrase activity upregulates nrdF1, nrdF2, nrdH, and nrdI, potentially implicating NrdF1 in the provision of dNTPs for DNA repair or turnover (6). The dashed arrows denote the induction of expression of RNR-encoding genes under various stresses. Putative RNRs are followed by question marks.

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Bacterial strains and culture conditions. The bacterial strains and plasmids used in this study are detailed in Table 1. All Escherichia coli strains were grown in Luria-Bertani broth or on Luria agar. Unless otherwise indicated, M. smegmatis strains were grown in Middlebrook 7H9 medium (Merck) supplemented with 0.085% NaCl, 0.2% glucose, 0.2% glycerol, and 0.05% Tween 80 or on solid Middlebrook 7H10 medium supplemented with 0.085% NaCl, 0.2% glucose, and 0.5% glycerol. M. tuberculosis strains were grown in Middlebrook 7H9 medium supplemented with 0.2% glycerol, Middlebrook oleic acid-albumin-dextrose-catalase enrichment (Merck), and 0.05% Tween 80. Ampicillin (Ap) and kanamycin (Km) were used in E. coli cultures at final concentrations of 200 and 50 µg/ml, respectively; hygromycin (Hyg), Km, and gentamicin (Gm) were used in mycobacterial cultures at final concentrations of 50, 25, and 10 µg/ml, respectively. Where applicable, rifampin (rifampicin) (Rif) was used in M. smegmatis cultures at a concentration of 200 µg/ml.

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TABLE 1. Strains and plasmids used in this study

Allelic exchange mutagenesis and complementation of mutant strains. To create a suicide substrate for deletion mutagenesis of the nrdF1 gene in M. tuberculosis, an 8,467-bp fragment from bacterial artificial chromosome Rv420 carrying this gene was cloned into pGEM3Z(+)f. A 1,990-bp BamHI-MfeI fragment from this vector containing 39 bp of the 5' end of nrdF1 and a 2,481-bp Asp718-SnaBI fragment containing 47 bp of the 3' end of nrdF1 were then subcloned into pGEM3Z(+)f to create pGNRDF1. This vector contained the nrdF1 gene carrying an internal, out-of-frame, 883-bp deletion flanked by homologous sequences. A 4,483-bp EcoRI/BamHI fragment from pGNRDF1 was cloned into p2NIL before insertion of the lacZ-sacB-hyg cassette from pGOAL19 (46) to create p2TBF1KO. Suicide plasmids for the knockout of nrdB and nrdR in M. tuberculosis and M. smegmatis and nrdF2 in M. smegmatis were constructed by PCR amplification from genomic DNA of upstream and downstream homologous sequences including the 5' and 3' termini of the gene of interest by using the primer pairs described in Table S1 in the supplemental material. Amplicons were directly cloned into pGEM3Z(+)f, pGEM-T Easy (Promega), or pCR2.1-TOPO (Invitrogen) and were sequenced before the corresponding upstream and downstream fragments were subcloned into p2NIL to create out-of-frame deletions of the genes of interest. In some cases, the Hyg resistance cassette (hyg) from pIJ963 (2) was inserted at the junction site of the up- and downstream fragments to create a hyg-marked deletion allele. The lacZ-sacB cassette from pGOAL17 or the lacZ-sacB-hyg cassette from pGOAL19 was then inserted into the p2NIL subclones to create p2TBBKO and p2TBRKO as allelic exchange substrates for introducing unmarked deletions in the M. tuberculosis nrdB and nrdR genes, respectively, and p2SMF2KO and p2SMRKO as substrates for introducing hyg-marked deletions in the M. smegmatis nrdF2 and nrdR genes, respectively (Table 1). Suicide vectors were electroporated into M. tuberculosis H37Rv or M. smegmatis mc²155, and allelic exchange mutants were recovered by two-step selection, as previously described (25, 46).

Vectors pNRDF2, which carries the M. tuberculosis nrdF2 gene (16), and pNRDR, which carries the M. smegmatis nrdR gene, were used for genetic complementation of M. smegmatis nrdF2 and nrdR mutants, respectively (Table 1). pNRDR was constructed by PCR amplification of M. smegmatis genomic DNA by using the primers described in Table S2 in the supplemental material to produce a 967-bp fragment containing nrdR and flanking sequences that was cloned as an Asp718-HindIII fragment into pMV306K.

Testing of susceptibilities of mycobacterial strains. The susceptibilities of mycobacterial strains to mitomycin C (MTC) and HU were determined by plating serial dilutions of log-phase cultures (optical density at 600 nm [OD₆₀₀], 0.6) onto Middlebrook 7H10 medium containing either HU, at concentrations up to 80 mM (6,084 µg/ml), or MTC (0 to 0.1 µg/ml) and enumerating CFU after incubation at 37°C. M. smegmatis plates containing MTC were scored after 4 to 7 days of incubation; those containing HU were scored after 4 (3 mM HU), 14 (6 to 20 mM HU), or 28 (>20 mM HU) days. M. tuberculosis plates were scored after 8 weeks of incubation with MTC or after 3, 5, or 12 weeks of incubation with HU at 3, 6, or 9 mM, respectively. Survival of M. tuberculosis in the presence of acidified nitrite was determined as described by Firmani and Riley (20). Briefly, mid-logarithmic-phase cultures (OD₆₀₀, 0.6) were diluted 1:10 and incubated for 24 h in Middlebrook 7H9 medium (pH 5.3) supplemented with NaNO₂ at concentrations up to 48 mM before serial dilutions were plated for CFU enumeration. Strain survival after UV irradiation was assessed by plating serial dilutions of logarithmic-phase cultures onto Middlebrook 7H10 medium and then exposing the open plates to UV irradiation in a Stratalinker 1800 cross-linker (0 to 40 mJ/cm²). The MICs of HU, MTC, ofloxacin (OFX), novobiocin (NVB), and streptomycin (STR) were determined by broth microdilution (17).

Mutagenesis assays. The rates of spontaneous mutation of M. smegmatis strains to Rif resistance were determined by Luria-Delbrück fluctuation analysis (38, 54), and frequencies of UV-induced mutation to Rif resistance were determined as previously described (7).

Gene expression analysis by real-time qRT-PCR. RNA was extracted from early-logarithmic-phase cultures by previously described methods (18). Primers for real-time quantitative reverse transcription-PCR (qRT-PCR) analysis of the expression of the M. smegmatis and M. tuberculosis nrdB, nrdE, and nrdF2 genes, M. tuberculosis nrdF1, and M. smegmatis sigA were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and are described in Table S2 in the supplemental material. The expression levels of M. tuberculosis sigA were determined using the primers described by Dawes et al. (16). The synthesis of cDNA and subsequent amplification with the LightCycler FastStart DNA Master Sybr green I kit in the Roche LightCycler (version 1.5) was carried out as previously described (35). Absolute numbers of transcripts were normalized to the number of sigA transcripts in the same sample, and where indicated, the normalized data were compared with normalized transcript levels in the wild-type (M. tuberculosis H37Rv or M. smegmatis mc²155) control.

Mouse infections. Eight- to 10-week-old female B6D2/F₁ mice from Jackson Laboratories (Bar Harbor, ME) were infected by exposure to aerosol particles in a nose-only infection apparatus (In Tox Products, Albuquerque, NM), which resulted in the seeding of 1.3 to 2.3 log₁₀ bacteria within the mouse lungs (41). Three mice were sacrificed per time point over a period of 126 days. The lungs, livers, and spleens of infected animals were harvested and homogenized, and serial dilutions were plated in order to enumerate organ bacterial loads.

Statistics. The independent Student t test or an unpaired t test was used to determine the statistical significance of pairwise comparisons using GraphPad Prism software.

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Loss of the alternate R2-encoding genes has no effect on the growth, drug susceptibility, or virulence of M. tuberculosis. Unmarked mutants of M. tuberculosis H37Rv carrying targeted deletions of the nrdF1 or nrdB gene were constructed by allelic exchange (46). Neither the nrdF1 nor the nrdB mutant strain displayed a growth phenotype when cultured aerobically in Middlebrook 7H9 medium (data not shown). The strains were then tested for sensitivity to (i) the class I RNR inhibitor HU, (ii) inhibitors of metabolism that have been shown to induce specific nrd genes in M. tuberculosis (OFX, NVB, and STR) (6), and (iii) genotoxic stress caused by MTC, an inducer of nrdF2 expression (48), or by UV irradiation, a potent inducer of the SOS response (7). Sensitivities to compounds were tested by MIC determination (Table 2) and, in the case of HU and MTC, also by the use of a more sensitive plating assay (Fig. 3).

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TABLE 2. MICs of various antibiotics and compounds for parentala and nrd mutant strains of M. tuberculosis and M. smegmatis

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FIG. 3. Growth and survival of the nrdF1 and nrdB mutants of M. tuberculosis in vitro and in vivo. (A and B) Plating assays for survival of mutant strains in the presence of HU (A) or MTC (B). Logarithmic-phase cultures were plated onto Middlebrook 7H10 agar supplemented with HU or MTC, and growth of bacteria was assessed by scoring CFU, as described in Materials and Methods. (C) Survival of the nrdB mutant upon exposure to acidified nitrite. Bacteria were exposed to different concentrations of acidified NaNO2 for 24 h, followed by plating on solid medium in order to score survival. Data in panels A, B, and C are averages and standard deviations from three biological replicates. (D) Growth of the nrdF1, nrdB, and wild-type strains in the lungs of B6D2/F1 mice. Mice were infected aerogenically, and the bacillary loads in the lungs of the infected animals were determined by CFU assessment over a period of 126 days. Each data point represents the mean for three mice per group. Error bars, standard deviations. , H37Rv; , nrdF1 strain; , nrdB strain.

The HU and MTC sensitivities of the nrdF1 and nrdB mutants were indistinguishable from those of the wild type in both assays (Fig. 3A and B; Table 2). The mutants also showed no difference in sensitivity to UV irradiation from the wild type (data not shown). Furthermore, loss of nrdF1 or nrdB gene function had no discernible effect on the susceptibility of M. tuberculosis to OFX, NVB, or STR (Table 2). To determine whether loss of the putative class Ic RNR small subunit, NrdB, affected the sensitivity of M. tuberculosis to nitrosative stress, the nrdB mutant and wild-type strains were exposed to increasing concentrations of acidified nitrite before being plated onto Middlebrook 7H10 agar in order to monitor survival. However, no differential susceptibility was detected for the mutant strain (Fig. 3C).

The effect of alternate R2-encoding gene loss on the virulence of M. tuberculosis was then assessed by testing the abilities of the nrdF1 and nrdB mutant strains to grow in mouse lungs after aerosol infection. Both strains were able to grow logarithmically during the first 4 weeks of infection, and both established a stable steady state at high bacillary loads, with kinetics and organ bacillary loads similar to those of the wild-type strain (Fig. 3D). Furthermore, no differences were noted in the gross pathology of the lungs or in the kinetics or extent of bacterial hematogenous dissemination to the spleen and liver (data not shown).

Quantitative analysis of RNR-encoding nrd gene expression levels in wild-type and nrd mutant strains of M. tuberculosis. To establish the expression levels of the various nrd mRNAs, relative to the sigA internal gene expression control, in wild-type and mutant M. tuberculosis strains, comparative transcript levels were determined by real-time qRT-PCR during early-logarithmic-phase growth. The nrdE gene served as the target sequence for the nrdHIE gene cluster, which is likely to constitute an operon (Fig. 1) (50). The nrdE and nrdF2 genes were expressed at levels comparable to one another during this phase of growth (Table 3). In contrast, the levels of expression of nrdF1 and nrdB were considerably lower than that of nrdF2 (four- and sixfold, respectively), and nrdZ was expressed at an even lower level under the conditions tested (Table 3).

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TABLE 3. Normalized levels of nrd gene transcripts in mycobacteria during early-logarithmic-phase aerobic growth in Middlebrook 7H9 medium

We then investigated the effects of nonessential nrd gene loss on the expression of the remaining nrd genes (Table 4). Deletion of nrdF1 did not have a significant effect on the expression of nrdE, nrdF2, nrdB, or nrdZ in M tuberculosis. Similarly, the expression of nrdE, nrdF1, nrdF2, and nrdZ was unaffected by loss of nrdB, while loss of nrdZ had no effect on the expression of nrdE, nrdF1, nrdF2, or nrdB. Together, these observations argued against regulatory cross talk between the three R2-encoding genes or between the class I and class II RNR-encoding genes under the conditions tested. However, the possibility of regulatory cross talk under different conditions could not be excluded.

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TABLE 4. Real-time qRT-PCR analysis of nrd gene expression in mycobacterial strains

The class Ib RNR is essential in M. smegmatis. M. smegmatis mc²155 contains a large, IS1096-flanked genomic duplication that carries, among others, the nrdHIE operon and the nrdF2 gene, which encode the class Ib RNR (Fig. 1) (64). One copy of the nrdF2 gene could be readily inactivated in mc²155 to produce a mutant carrying both wild-type nrdF2 and mutant nrdF2::hyg alleles (see Fig. S1 in the supplemental material). In contrast, all attempts to insertionally inactivate nrdF2 in the DRKIN strain, a laboratory derivative of mc²155 that lacks the 56-kb chromosomal duplication (64), proved unsuccessful. However, double-crossover recombinants in which the endogenous nrdF2 gene was replaced by an nrdF2::hyg allele were readily obtained when a complementing copy of the homologous nrdF2 gene from M. tuberculosis (16) was integrated at the attB site (see Fig. S1 in the supplemental material). Therefore, as in M. tuberculosis (16), the class Ib RNR, NrdEF2, is essential in M. smegmatis. Moreover, nrdB could not substitute for the function of the nrdF2 gene under the conditions tested, even though nrdB is expressed in M. smegmatis (Table 3).

Consistent with the genotype, the transcript levels of nrdE and nrdF2 in the DRKIN mutant were 50% lower than those observed in mc²155 (Tables 3 and 4). Furthermore, insertional inactivation of one copy of nrdF2 in mc²155 halved the relative expression of this gene only (Tables 3 and 4); the expression of nrdB and nrdE was unchanged in the nrdF2::hyg mutant (Table 4).

NrdR regulates the transcription of the essential class Ib RNR-encoding genes in mycobacteria. NrdR was recently identified as a regulator of bacterial nrd gene transcription (4, 53, 60) Homologues of the putative NrdR in M. tuberculosis H37Rv (Rv2718c) are present in all sequenced mycobacterial species, including M. leprae. In mycobacteria, nrdR is proximal to the lexA gene, but, unlike the nrdR gene in Streptomyces coelicolor, it does not colocalize with any RNR-encoding genes (Fig. 4A). Mycobacterial NrdRs show a high degree of homology to Streptomyces coelicolor NrdR, with all critical residues conserved, including the zinc ribbon and the ATP cone domains (Fig. 4B). Canonical NrdR boxes have been identified upstream of nrdH and nrdF2 in mycobacteria (Fig. 4C and D) (53) but are not found upstream of nrdB, nrdF1, or nrdZ in any of the sequenced mycobacterial genomes harboring one or more of these genes.

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FIG. 4. Homology and synteny of nrdR genes in mycobacteria. (A) The nrdR gene in S. coelicolor is located upstream of nrdJ, but this genetic organization is not conserved in mycobacteria, where nrdR is associated with other genes of unrelated function. Homologous genes are depicted as arrows with the same shading. (B) The NrdR proteins from M. tuberculosis and M. smegmatis show a high degree of homology to the corresponding repressor of nrd gene expression in S. coelicolor (4), with the zinc ribbon and ATP cone (bold) domains all highly conserved between these species. (C and D) Putative NrdR boxes (boldfaced) located upstream of the nrdHIE gene cluster and the nrdF2 gene, respectively, identified on the basis of the consensus palindromic sequence acaCwAtATaTwGtgt (uppercase letters represent highly conserved nucleotides) (53).

To investigate the role of NrdR in the regulation of RNR-encoding genes in mycobacteria, nrdR was inactivated in M. tuberculosis and M. smegmatis to produce unmarked (nrdR) and hyg-marked (nrdR::hyg) mutant strains, respectively. Elimination of nrdR function resulted in a significant increase in the expression of nrdE and nrdF2 in both M. tuberculosis and M. smegmatis during early-logarithmic-phase growth but had no effect on the expression of nrdB, nrdF1, or nrdZ in M. tuberculosis (Table 4). Conversely, complementation of the M. smegmatis nrdR::hyg mutant reduced the expression of nrdE and nrdF2 almost to wild-type levels (data not shown), demonstrating that, in mycobacteria, NrdR regulates the expression of the essential class Ib RNR-encoding genes only (Table 4).

Effects of altered expression of class Ib RNR-encoding genes on mutagenesis and the susceptibility of mycobacteria to HU and genotoxic stress. The phenotypic consequences of the possible changes in RNR activity arising from altered expression of class Ib RNR-encoding genes were then assessed. Interestingly, the nrdF2::hyg mutant of M. smegmatis displayed a significant increase in sensitivity to HU over that of its parent, mc²155, as evidenced by the 2.1 log₁₀-fold reduction in the CFU formation of this mutant on plates containing 9 mM HU (Fig. 5A) (P < 0.0001) and a concomitant two- to fourfold reduction in the MIC (Table 2). Complementation of the nrdF2::hyg mutant with M. tuberculosis nrdF2 resulted in partial restoration of HU sensitivity to wild-type levels (Fig. 5A), strongly implicating the loss of a copy of nrdF2 in the HU-hypersensitive phenotype of this strain. The incomplete restoration of HU sensitivity could result from complementation with a heterologous gene that may not be equivalent to M. smegmatis nrdF2 in terms of expression and function. In contrast, the susceptibility of the nrdF2::hyg mutant to both MTC and UV irradiation was indistinguishable from that of its parent, with both strains displaying markedly greater resistance to UV damage than a UV-hypersensitive control lacking the dnaE2 gene (7) (Fig. 5B and C). As such, the hypersensitivity of the nrdF2::hyg mutant was restricted to HU. The DRKIN mutant also displayed marked hypersensitivity to HU relative to mc²155 (Fig. 5A [P < 0.001 at 9 mM HU]; Table 2). However, unlike the nrdF2::hyg mutant, which was significantly hypersensitive to HU but not to MTC or to UV irradiation (Fig. 5), the DRKIN strain—which has a reduced dosage of numerous genes in addition to those encoding the class Ib RNR (64)—was also hypersensitive to MTC in both assays (Fig. 4B [P < 0.005 at MTC concentrations above 0.01 µg/ml]; Table 2), as well as to OFX and NVB in the plating assay (data not shown). However, this strain was not hypersensitive to UV irradiation (Fig. 5C).

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FIG. 5. Effects of altered expression of class Ib RNR-encoding genes on the susceptibilities of M. smegmatis to HU and genotoxic stress. (A and B) Survival in the presence of HU (A) or MTC (B). Logarithmic-phase cultures were serially diluted and plated onto Middlebrook 7H10 agar supplemented with HU or MTC, and growth was assessed by scoring CFU. (C) Survival after exposure of bacteria on solid medium to UV irradiation. , mc2155; , nrdF2::hyg strain; , nrdF2::hyg::pNRDF2; , DRKIN; *, dnaE2::aph strain. Data are averages and standard deviations from three biological replicates.

In contrast to the HU hypersensitivity conferred by reduced expression of nrdF2 alone (nrdF2::hyg) or together with nrdHIE (DRKIN), equivocal results were obtained when the HU susceptibility of the nrdR::hyg strain, in which nrdE and nrdF2 gene expression was elevated three- to fivefold over wild-type levels, was compared to that of mc²155. In some experiments, a small (ca. fivefold) increase in CFU was observed for the nrdR::hyg mutant at higher HU concentrations (40 to 80 mM), but, though reproducible, this difference was not statistically significant (data not shown). The nrdR mutant of M. tuberculosis also showed some evidence of increased resistance to HU in the plating assay, but again, this difference was not significant. Furthermore, neither nrdR mutant showed an increase in the HU MIC over that for its corresponding parental strain (Table 2).

Finally, since imbalances in dNTP pools have been shown to confer mutagenic effects on other organisms (24, 66), we analyzed the rates of spontaneous mutation to Rif resistance in the nrdF2::hyg, DRKIN, and nrdR::hyg mutants and their parental wild-type strains. All strains showed similar mutation rates (probabilities of 4.4 x 10^–9, 6.3 x 10^–9, and 8.2 x10^–9 mutation per cell per generation for the nrdF2::hyg, DRKIN, and nrdR::hyg mutants versus 5.7 x 10^–9 mutation per cell per generation for M. smegmatis mc²155). Moreover, no differences were observed in the frequencies of UV irradiation-induced mutation to Rif resistance; the DRKIN, nrdR::hyg, and mc²155 strains showed comparable levels of induced mutation that were markedly higher than that of the dnaE2 deletion mutant, which served as an induced-mutagenesis-defective control (7) (see Fig. S2 in the supplemental material).

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The recent identification and characterization of the R2 subclass found in Chlamydia (27) suggested that the class Ic RNR may represent an adaptation of the enzyme that confers increased resistance to poisoning by reactive nitrogen and oxygen intermediates, such as NO (21, 30), to which intracellular pathogens are exposed during the course of infection. The class Ic R2 is the only small RNR subunit found in Chlamydia and other organisms, and it associates with a class Ia-type large subunit (NrdA) to form a functional enzyme (27). In contrast, mycobacteria also contain at least one class Ib R2 subunit (NrdF2 and, in some cases, NrdF1) in addition to the class Ic R2 (NrdB) (Fig. 1 and 2) but contain only a class Ib-type large subunit (NrdE). In prior work, we demonstrated the essentiality of nrdF2 for aerobic growth of M. tuberculosis in vitro, which suggested that the alternate class I R2s, NrdB and NrdF1, are unable to substitute for NrdF2 and also that the class II RNR, NrdZ, could not substitute for NrdEF2 under the conditions tested (16).

In the present study, we investigated the functions of the nrdB and nrdF1 genes in M. tuberculosis by analyzing the consequences of targeted disruption of these genes for the growth and survival of the organism in vitro under conditions predicted to be physiologically revealing—nitrosative stress in the case of nrdB (27) and translational or genotoxic stress in the case of nrdF1 (6)—as well as for growth and survival in a mouse infection model. The central roles of NO in controlling the growth of M. tuberculosis in mice and in modulating the metabolism and physiology of the organism after activation of the acquired immune response are well established in this model of infection (11, 12, 43, 55). It would seem intuitive, therefore, that during the chronic stage of infection, when immune-mediated nitrosative assault is at its peak, M. tuberculosis is most likely to utilize enzymes, such as the putative class Ic RNR, that resist poisoning by NO. Importantly, however, both nrdB and nrdF1 mutant strains were indistinguishable from the wild type under all conditions tested. The dispensability of nrdB for survival during chronic infection suggests that the need for RNR-catalyzed production of dNTPs under the conditions of limited chromosomal replication that are thought to prevail at this stage of infection (42) can be met by the class Ib RNR, which also provides the dNTPs for replication during acute infection. Similarly, despite the transcriptional upregulation of nrdF1 that occurs in response to inhibition of translation or DNA gyrase activity (6), deletion of this gene has no effect on the susceptibility of M. tuberculosis to STR, OFX, or NVB or on its growth and survival in the mouse lung. In the case of NrdF1, the relative weakness of the interaction of this alternative R2 subunit with NrdE (69) may restrict its ability to compete with NrdF2 for binding to the R1 subunit. Similarly, both the ability of the nrdB-encoded R2 subunit to form a catalytically active class Ic RNR with NrdE and the relative strength of the putative interaction between NrdB and NrdE have yet to be established. However, the notion that competitive binding to NrdE may play a key role in determining the contribution of the various R2 subunits to overall RNR activity in M. tuberculosis is supported by our expression data: specifically, nrdE transcript levels argue against the availability of surplus levels of the large subunit for interaction with the alternate R2s, which are expressed at moderately lower levels than NrdF2.

A regulatory association between nrdHIE and nrdF2, which did not extend to the other RNR-encoding genes found in these organisms, was also observed in mycobacteria. In particular, NrdR was shown specifically to repress nrdHIE and nrdF2 in M. tuberculosis and M. smegmatis, as evidenced by the marked increases in the levels of nrdF2 and nrdE transcripts in nrdR-deficient mutants. In contrast, the expression of nrdB in both mycobacterial species, and that of nrdF1 and nrdZ in M. tuberculosis, was unaffected by a loss of NrdR function. This finding is consistent with the lack of identifiable NrdR boxes upstream of these genes and differentiates mycobacteria from other organisms in which the function of the NrdR regulator has been investigated. In E. coli, for example, NrdR negatively regulates the expression all three classes of RNRs, although deletion of the nrdR gene has a much greater effect on expression of the class Ib RNR genes (nrdHIEF) than on that of the class Ia (nrdAB) or class III (nrdDG) genes (60). In S. coelicolor, nrdR regulates both the class II RNR-encoding nrdJ gene, with which it is operonic (Fig. 3), and the nrdABS operon (4). As in E. coli, these sets of RNR-encoding genes were differentially affected by NrdR loss, but in this case, nrdJ was more highly induced than nrdABS (4). In Streptomyces, a further level of regulation exists in the form of a riboswitch that represses nrdAB expression in the presence of vitamin B₁₂ (3). Although M. tuberculosis also contains a putative vitamin B₁₂-dependent RNR (NrdZ), no B₁₂ riboswitches were identified upstream of other RNR-encoding genes (65), suggesting that vitamin B₁₂ does not regulate RNR gene expression in this organism. The specific signals that lead to derepression of the nrdR-regulated nrdHIE and nrdF2 genes in mycobacteria have yet to be established. However, they must differ from the signals that result in the coinduction of these genes along with nrdF1, which is triggered by inhibition of translation or DNA gyrase function (6).

The mutant strains of M. smegmatis described here and in a previous study (64) provided a means of assessing the phenotypic effects of altered expression of class Ib RNR-encoding genes in mycobacteria. Recapitulation of the HU hypersensitivity of the DRKIN mutant by inactivating one of the duplicated copies of nrdF2 in M. smegmatis mc²155 directly implicated the dosage of class Ib RNR-encoding genes in this phenotype. This observation confirms that NrdEF2 is the principal target for HU in mycobacteria and provides a good example of the use of target knockdown to probe the specificity of inhibitors in a whole-cell assay. Loss of NrdR function resulted in overexpression of nrdHIE and nrdF2 in M. tuberculosis and M. smegmatis, but this effect did not translate into a significant increase in resistance to HU. This observation contrasts with findings in other systems in which overproduction of class I RNR leads to increased resistance to HU (13, 23, 29, 56). In a further departure from other systems (9, 10, 24, 26, 66), induction of the class Ib RNR in M. smegmatis by derepression of nrdHIE and nrdF2 did not affect growth or confer hypermutability. The reasons underlying these observations are unclear but may include the existence of allosteric and/or other mechanisms regulating RNR function (24) and dNTP pools in mycobacteria. The availability of improved methods to determine nucleotide concentrations directly (8) should allow variations in dNTP pools resulting from altered levels of mycobacterial RNR gene expression to be monitored and correlated with changes in the physiological state of these organisms.

The nrdF2::hyg mutant was specifically hypersensitive to HU. This strain, in which the nrdF2 gene dosage was halved, showed no increase in sensitivity to MTC, even though nrdF2 is induced by this compound in M. tuberculosis (48). In contrast, the hypersensitivity phenotype of the DRKIN mutant was not restricted to HU but extended to include genotoxic agents such as MTC and OFX. It is tempting to speculate that this differential phenotype is attributable to the halving in dosage of another gene(s) carried on the duplicated region of the mc²155 chromosome (64). One possible candidate in this regard is dinP, since this gene encodes a putative PolIV (DinB)-type, Y-family DNA polymerase whose orthologs in other organisms are involved in translesion synthesis across replication-blocking lesions (28). An investigation of the molecular basis of the generalized genotoxic stress hypersensitivity of the DRKIN strain, which includes an analysis of the role of dinP, is currently under way in our laboratory.

In conclusion, our results suggest that in the mouse model, NrdEF2 alone provides the RNR activity required by M. tuberculosis for DNA synthesis and repair at every stage of infection. Consequently, these findings argue against specialist roles for NrdZ, NrdF1, and NrdB under conditions of genotoxic and nitrosative stress encountered during the course of infection in mice, and thus they differentiate M. tuberculosis from organisms that utilize a multiplicity of RNRs to modulate the provision of dNTPs for DNA replication and repair under variable and hostile environmental conditions. Instead, our observations have revealed a potential vulnerability in dNTP provision in M. tuberculosis, thereby establishing a compelling rationale for the pursuit of the NrdEF2 form of RNR as a target for antitubercular drug discovery (45, 69).

 
This work was supported by an International Research Scholars grant from the Howard Hughes Medical Institute (to V.M.); by grants from the South African Medical Research Council (to V.M.), the National Research Foundation (to V.M. and M.B.M.), the National Institutes of Health (RO1 AI54338 and AI54361, to G.K), and the Columbia University-Southern African Fogarty AIDS International Research and Training Program (grant 5 D43 TW00231, FIC, NIH, to B.D.K and M.B.M.); and by a Mellon Postgraduate Mentoring Award from the University of the Witwatersrand (to M.B.M. and V.M).

We are grateful to Bhavna Gordhan, Nackmoon Sung, and Stephanie Dawes for advice and assistance and to Stewart Cole for providing the M. tuberculosis cosmid library.

 
* Corresponding author. Mailing address: Molecular Mycobacteriology Research Unit, National Health Laboratory Service, P.O. Box 1038, Johannesburg 2000, South Africa. Phone: 2711-4899030. Fax: 2711-4899397. E-mail for Bavesh D. Kana: bavesh.kana{at}nhls.ac.za. E-mail for Valerie Mizrahi: valerie.mizrahi{at}wits.ac.za

Published ahead of print on 21 November 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, February 2009, p. 985-995, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01409-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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