JOURNAL OF BACTERIOLOGY, Feb. 2009, p. 985–995
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 3
Function and Regulation of Class I Ribonucleotide
Reductase-Encoding Genes in Mycobacteria?†
Mohube B. Mowa,1Digby F. Warner,1Gilla Kaplan,2Bavesh D. Kana,1* and Valerie Mizrahi1*
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,1and 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
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.
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 Myco-
bacterium 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,
Ribonucleotide reductases (RNRs) play an essential role
in all living cells by catalyzing the reduction of ribonucleo-
side-5?-di- or triphosphates to generate the deoxyribonucle-
otides (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 require-
ments 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 re-
cently 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) co-
factor 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 resis-
tant to reactive nitrogen and oxygen species (27), including the
antimicrobial effector nitric oxide (NO), which targets the ty-
rosyl 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 mycobac-
teria reveals both common and unique features (Fig. 1) (http:
//rnrdb.molbio.su.se). All possess a class Ib RNR encoded by
* Corresponding author. Mailing address: Molecular Mycobacteri-
ology 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: firstname.lastname@example.org.
E-mail for Valerie Mizrahi: email@example.com.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 21 November 2008.
nrdE and a genetically linked nrdF gene, designated nrdF2
(Rv3048c in the reference organism, M. tuberculosis H37Rv
). nrdE is the only R1-encoding gene identified in myco-
bacteria. Species other than Mycobacterium leprae and Myco-
bacterium ulcerans also possess an R2 subunit-encoding gene
homologous to that of the chlamydial class Ic RNR (27), des-
ignated nrdB (Rv0233 in M. tuberculosis H37Rv ). M. tu-
berculosis 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). Mycobacte-
rium 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
Given the multiplicity of RNR-encoding genes in M. tu-
berculosis and their transcriptional responsiveness to differ-
ent 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 condi-
tions 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 condi-
tions 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 en-
zyme as a novel target for antitubercular drug discovery (16,
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.
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.
986 MOWA ET AL.J. BACTERIOL.
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 an-
other 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 en-
zyme, reinforcing the apparently dominant role of the class
Ib enzyme in nucleotide cycling and dNTP pool modulation
MATERIALS AND METHODS
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. smeg-
matis 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 kana-
mycin (Km) were used in E. coli cultures at final concentrations of 200 and 50
?g/ml, respectively; hygromycin (Hyg), Km, and gentamicin (Gm) were used in
mycobacterial cultures at final concentrations of 50, 25, and 10 ?g/ml, respec-
TABLE 1. Strains and plasmids used in this study
Strain or plasmid DescriptionSource
ATCC 25618; virulent laboratory strain
Derivative of H37Rv carrying an unmarked deletion in nrdF1
Derivative of H37Rv carrying an unmarked deletion in nrdB
Derivative of H37Rv carrying an unmarked deletion in nrdR
ept-1 (efficient plasmid transformation) mutant of mc26
Derivative of mc2155 carrying a hyg-marked deletion in nrdF2; Hygr
Derivative of the ?nrdF2::hyg strain carrying the M. tuberculosis nrdF2 gene
integrated at the attB locus (via pNRDF2); HygrGmr
Derivative of mc2155 lacking the 56-kb chromosomal duplication
Single-crossover recombinant between ?DRKIN and p2?SMF2KO; HygrKmr
Derivative of ?DRKINSCO carrying pNRDF2; HygrKmrGmr
Derivative of ?DRKIN carrying a hyg-marked deletion in the remaining
chromosomal copy of nrdF2 and the M. tuberculosis nrdF2 gene integrated at the
attB locus (via pNRDF2); HygrGmr
Derivative of mc2155 carrying a hyg-marked deletion in nrdR; Hygr
Derivative of the ?nrdR::hyg strain carrying M. smegmatis nrdR integrated at the
attB locus (via pNRDR); HygrKmr
Derivative of mc2155 carrying a dnaE2::aph allele; Kmr
E. coli cloning vector; Kmr
E. coli cloning vector; Apr
E. coli PCR TA cloning vector; Apr
E. coli PCR TA cloning vector; AprKmr
E. coli-Mycobacterium integrating shuttle vector; derivative of pHINT carrying a
Km resistance cassette; KmrApr
E. coli-Mycobacterium integrating shuttle vector; derivative of pMV306acarrying an
aph gene; Kmr
Plasmid carrying the lacZ and sacB genes as a PacI cassette; Apr
Plasmid carrying the lacZ, sacB, and hyg genes as a PacI cassette; Apr
Plasmid carrying the hyg resistance gene; AprHygr
Derivative of pGINT carrying the nrdF2 gene from M. tuberculosis; GmrApr
Derivative of pMV306K carrying the nrdR gene from M. smegmatis; Kmr
Derivative of pGEM3Z(?)f carrying an 883-bp deletion in the M. tuberculosis
nrdF1 gene and 3?- and 5?-flanking sequences; Apr
Knockout vector for creating an unmarked deletion in M. tuberculosis nrdF1; Kmr
Knockout vector for creating an unmarked deletion in M. tuberculosis nrdB; Kmr
Knockout vector for creating an unmarked deletion in M. tuberculosis nrdR; Kmr
Knockout vector for creating a hyg-marked deletion in M. smegmatis nrdF2; Kmr
Knockout vector for creating a hyg-marked deletion in M. smegmatis nrdR; Kmr
aSee reference 58.
VOL. 191, 2009RIBONUCLEOTIDE REDUCTASES IN MYCOBACTERIA987
tively. Where applicable, rifampin (rifampicin) (Rif) was used in M. smegmatis
cultures at a concentration of 200 ?g/ml.
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 ho-
mologous 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 p2?TBF1KO. 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 homol-
ogous 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 dele-
tions 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 p2?TBBKO and p2?TBRKO as allelic exchange
substrates for introducing unmarked deletions in the M. tuberculosis nrdB and
nrdR genes, respectively, and p2?SMF2KO and p2?SMRKO 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 ?g/ml), or MTC (0 to 0.1 ?g/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
NaNO2at 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), novo-
biocin (NVB), and streptomycin (STR) were determined by broth microdilution
Mutagenesis assays. The rates of spontaneous mutation of M. smegmatis
strains to Rif resistance were determined by Luria-Delbru ¨ck fluctuation analysis
(38, 54), and frequencies of UV-induced mutation to Rif resistance were deter-
mined 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 de-
scribed 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/F1mice 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 log10bacteria 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
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 in-
ducer 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
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 in-
TABLE 2. MICs of various antibiotics and compounds for parentala
and nrd mutant strains of M. tuberculosis and M. smegmatis
MIC (?g/ml) of the following antibiotic or
aThe parental strains were H37Rv and mc2155. MICs were determined in
988 MOWA ET AL.J. BACTERIOL.
creasing 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 vir-
ulence 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 differ-
ences were noted in the gross pathology of the lungs or in
the kinetics or extent of bacterial hematogenous dissemina-
tion 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 con-
stitute 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).
We then investigated the effects of nonessential nrd gene
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 NaNO2for 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/F1mice. 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; Œ, ?nrdF1 strain; f, ?nrdB strain.
VOL. 191, 2009RIBONUCLEOTIDE REDUCTASES IN MYCOBACTERIA 989
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 condi-
tions could not be excluded.
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 endog-
enous nrdF2 gene was replaced by an nrdF2::hyg allele were
readily obtained when a complementing copy of the homolo-
gous 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 essen-
tial 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 (Ta-
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 homol-
ogy to Streptomyces coelicolor NrdR, with all critical residues
conserved, including the zinc ribbon and the ATP cone do-
mains (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.
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.
TABLE 3. Normalized levels of nrd gene transcripts in mycobacteria during early-logarithmic-phase aerobic growth in
Middlebrook 7H9 medium
Expression level of the transcript of the following gene relative to that of sigAa:
nrdEnrdF2 nrdF1nrdB nrdZ
M. tuberculosis H37Rv
M. smegmatis mc2155
M. smegmatis ?DRKIN
5.2 ? 0.9
60 ? 3
38 ? 7
5.1 ? 0.2
17 ? 4
9.6 ? 3.6
1.4 ? 0.1
0.91 ? 0.34
5.5 ? 1.8
4.9 ? 1.0
0.13 ? 0.06
aExpression levels were measured in cultures at an OD600of 0.3. N/A, not applicable.
TABLE 4. Real-time qRT-PCR analysis of nrd gene expression in mycobacterial strains
Normalized expressionaof the following gene by the indicated strain relative to that by the wild type:
nrdEnrdF1 nrdF2nrdB nrdZ
0.7 ? 0.2
0.8 ? 0.3
1.1 ? 0.2
2.8 ? 0.4***
1.6 ? 0.7
2.1 ? 0.7
1.1 ? 0.2
3.1 ? 0.3**
1.2 ? 0.6 1.2 ? 0.6
1.1 ? 0.31.2 ? 0.6
0.9 ? 0.3
1.00 ? 0.03
1.1 ? 0.4
1.1 ? 0.2 1.2 ? 0.5
0.6 ? 0.1*
1.3 ? 0.2
4.9 ? 0.8***
0.6 ? 0.2*
0.5 ? 0.1*
3.7 ? 0.7**
1.0 ? 0.2
1.0 ? 0.2
0.7 ? 0.2
a?, P ? 0.1; ??, P ? 0.01; ???, P ? 0.001. Statistical significance is based on pairwise comparison, by the unpaired t test, of the sigA-normalized expression level of
the gene of interest in the mutant strain with that in its parental wild-type strain. N/A, not applicable.
990MOWA ET AL.J. BACTERIOL.
smegmatis ?nrdR::hyg mutant reduced the expression of nrdE
and nrdF2 almost to wild-type levels (data not shown), dem-
onstrating that, in mycobacteria, NrdR regulates the expres-
sion of the essential class Ib RNR-encoding genes only
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 pos-
sible changes in RNR activity arising from altered expression
of class Ib RNR-encoding genes were then assessed. Interest-
ingly, 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
Rv2717c Rv2716 Rv2715
M. smegmatis CCTGGGAATTTCAGAAATGTTATTCAGAACATCTTGTATGGCTTCTTC-TGTGG------
M. smegmatis -------GGCCACTAGGTGTAGTGTCTGAGAG-ACCGACAGGCCACCACAGTTCGGGAGC
** ************* * * *** *** * * * *
** ********* ** *** *** ************* **** **
M. smegmatis GGTGAAACGCCACGTCGCGCTTGCGGAG----TTCGCGTGTGAACGCGACACGCCCAACC
M. smegmatis ACAACATCTCGGGGAGCGCCGCGAACTTTCCCACCAGTTGTAGTGTT
* * * * * ** * * * *
Zinc ribbon domain
ATP cone domain
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).
VOL. 191, 2009RIBONUCLEOTIDE REDUCTASES IN MYCOBACTERIA991
?nrdF2::hyg mutant with M. tuberculosis nrdF2 resulted in par-
tial 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 resto-
ration 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 con-
trast, the susceptibility of the ?nrdF2::hyg mutant to both MTC
and UV irradiation was indistinguishable from that of its par-
ent, 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). How-
ever, unlike the nrdF2::hyg mutant, which was significantly hy-
persensitive to HU but not to MTC or to UV irradiation (Fig.
5), the ?DRKIN strain—which has a reduced dosage of nu-
merous 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 ?g/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).
In contrast to the HU hypersensitivity conferred by re-
duced 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 ob-
served 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 mu-
tant 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 ana-
lyzed the rates of spontaneous mutation to Rif resistance in the
?nrdF2::hyg, ?DRKIN, and ?nrdR::hyg mutants and their pa-
rental wild-type strains. All strains showed similar mutation
rates (probabilities of 4.4 ? 10?9, 6.3 ? 10?9, and 8.2 ?10?9
mutation per cell per generation for the ?nrdF2::hyg, ?DR-
KIN, and ?nrdR::hyg mutants versus 5.7 ? 10?9mutation per
cell per generation for M. smegmatis mc2155). Moreover, no
differences were observed in the frequencies of UV irradi-
ation-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
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; Œ, ?nrdF2::hyg::pNRDF2; E, ?DRKIN; ?, dnaE2::aph strain. Data are averages and standard
deviations from three biological replicates.
992MOWA ET AL.J. BACTERIOL.
dnaE2 deletion mutant, which served as an induced-mutagen-
esis-defective control (7) (see Fig. S2 in the supplemental ma-
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 ox-
ygen 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 essenti-
ality 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 con-
sequences of targeted disruption of these genes for the growth
and survival of the organism in vitro under conditions pre-
dicted 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. tuber-
culosis is most likely to utilize enzymes, such as the putative
class Ic RNR, that resist poisoning by NO. Importantly, how-
ever, both nrdB and nrdF1 mutant strains were indistinguish-
able from the wild type under all conditions tested. The dis-
pensability of nrdB for survival during chronic infection
suggests that the need for RNR-catalyzed production of
dNTPs under the conditions of limited chromosomal replica-
tion 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 re-
sponse 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 sur-
vival 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 in-
teraction 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 sup-
ported 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 ex-
pressed 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 partic-
ular, 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 expres-
sion of the class Ib RNR genes (nrdHIEF) than on that of the
class Ia (nrdAB) or class III (nrdDG) genes (60). In S. coeli-
color, 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 fur-
ther 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 B12riboswitches were identified
upstream of other RNR-encoding genes (65), suggesting that
vitamin B12does 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 sig-
nals 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 pheno-
typic effects of altered expression of class Ib RNR-encoding
genes in mycobacteria. Recapitulation of the HU hypersensi-
tivity of the ?DRKIN mutant by inactivating one of the dupli-
cated copies of nrdF2 in M. smegmatis mc2155 directly impli-
cated the dosage of class Ib RNR-encoding genes in this
phenotype. This observation confirms that NrdEF2 is the prin-
cipal target for HU in mycobacteria and provides a good ex-
ample 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. tuber-
culosis 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 overproduc-
tion 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 dere-
pression of nrdHIE and nrdF2 did not affect growth or confer
hypermutability. The reasons underlying these observations
VOL. 191, 2009RIBONUCLEOTIDE REDUCTASES IN MYCOBACTERIA 993
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
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 con-
trast, 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 mo-
lecular basis of the generalized genotoxic stress hypersensitiv-
ity 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. tu-
berculosis for DNA synthesis and repair at every stage of in-
fection. Consequently, these findings argue against specialist
roles for NrdZ, NrdF1, and NrdB under conditions of geno-
toxic 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.
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