JOURNAL OF BACTERIOLOGY, Apr. 2010, p. 2220–2227
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 8
Role of the DinB Homologs Rv1537 and Rv3056 in
Bavesh D. Kana,1* Garth L. Abrahams,1Nackmoon Sung,1,2¶ Digby F. Warner,1Bhavna G. Gordhan,1
Edith E. Machowski,1Liana Tsenova,2James C. Sacchettini,3Neil G. Stoker,4
Gilla Kaplan,2and Valerie Mizrahi1*
MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research,
School of Pathology of the University of the Witwatersrand and the National Health Laboratory Service, Johannesburg,
South Africa1; Laboratory of Mycobacterial Immunity and Pathogenesis, Public Health Research Institute,
International Center for Public Health, 225 Warren St., Newark, New Jersey 07103-35352;
Texas A&M University, College Station, Texas 778433; and Royal Veterinary College,
London NW1 0TU, United Kingdom4
Received 26 August 2009/Accepted 28 January 2010
The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria
tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS)
across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA
polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and
survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-
dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses
revealed that DinB1, but not DinB2, interacts with the ?-clamp, consistent with its canonical C-terminal
?-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M.
tuberculosis to compounds that form N2-dG adducts and alkylating agents. Similarly, deletion of these genes
individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the
spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse
macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in
Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or
dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M.
tuberculosis do not behave like their counterparts from other organisms.
The emergence and global spread of multi- and extensively
drug-resistant strains of Mycobacterium tuberculosis have fur-
ther complicated the already daunting challenge of controlling
tuberculosis (TB) (15). The mechanisms that underlie the evo-
lution of drug resistance in M. tuberculosis by chromosomal
mutagenesis and their association with the conditions that tu-
bercle bacilli encounter during the course of infection are
poorly understood (6). It has been postulated that hypoxia, low
pH, nutrient deprivation, and nitrosative and oxidative stress
impose environmental and host immune-mediated DNA-dam-
aging insults on infecting bacilli (64). In addition, the observed
importance of excision repair pathways for the growth and
survival of M. tuberculosis in murine models of infection (13,
55) and the upregulation of M. tuberculosis genes involved in
DNA repair and modification in pulmonary TB in humans
provide compelling evidence that the in vivo environment is
DNA damaging (51).
Damage tolerance constitutes an integral component of an
organism’s response to genotoxic stress, preventing collapse of
the replication fork at persisting, replication-blocking lesions
through the engagement of specialized DNA polymerases that
are able to catalyze translesion synthesis (TLS) across the sites
of damage (19, 21, 60). Most TLS polymerases belong to the Y
family, which comprises a wide range of structurally related
proteins present in bacteria, archaea, and eukaryotes (44). Of
these, the DinB subfamily of Y family polymerases, whose
founder member is Escherichia coli Pol IV (63), is conserved
among all domains of life (44). The association of Y family
polymerases with inducible mutagenesis has implicated these
enzymes in the adaptation of bacteria to environmental stress
(17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified
in E. coli Pol IV: the polymerase catalyzes efficient and accu-
rate TLS across certain N2-dG adducts (27, 28, 34, 40, 45, 67)
and has been implicated in the tolerance of alkylation damage
(4); furthermore, overexpression of Pol IV significantly in-
creases mutation rates in E. coli (reviewed in references 21 and
26), and dinB is the only SOS-regulated gene required at in-
duced levels for stress-induced mutagenesis in this organism
(20). Furthermore, overproduction of E. coli Pol IV inhibits
replication fork progression through replacement of the repli-
cative polymerase to form an alternate replisome in which Pol
IV modulates the rate of unwinding of the DnaB helicase (25)
and also reduces colony-forming ability (61).
The M. tuberculosis genome encodes two Y family polymer-
ase homologs belonging to the DinB subfamily, designated
* Corresponding author. Mailing address: Molecular Mycobacteri-
ology Research Unit, National Health Laboratory Service, P.O. Box
1038, Johannesburg 2000, South Africa. Phone: 2711-4899370. Fax:
2711-4899397. E-mail for B.D.K.: email@example.com. E-mail for
¶ Present address: National Masan TB Hospital, Masan, South Korea.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 5 February 2010.
herein as DinB1 (DinX, encoded by Rv1537) and DinB2
(DinP, encoded by Rv3056), as well as a third, distantly related
homolog encoded by Rv3394c (see Fig. S1 in the supplemental
material) (9). On the basis of sequence similarity with their
counterparts from E. coli (63) and Pseudomonas aeruginosa
(54), including the complete conservation of key acidic resi-
dues essential for catalysis, DinB1 and DinB2 may be func-
tional DNA polymerases (see Fig. S1). In contrast, Rv3394c
lacks these residues and as such is unlikely to have polymerase
activity (see Fig. S1). Unlike most Y family polymerase-encod-
ing genes investigated with other bacteria (17, 26, 54, 58),
dinB1 and dinB2 expression in M. tuberculosis is not dependent
on RecA, the SOS response, or the presence of DNA damage
(5, 7, 52). That these genes are regulated by other mechanisms
and so may serve distinct roles in DNA metabolism in M.
tuberculosis is suggested by the observation that dinB1 is dif-
ferentially expressed in pulmonary TB (51) and is a member of
the SigH regulon (30), whereas expression of dinB2 is induced
following exposure to novobiocin (5).
In this study, we adopted a genetic approach to investigate
the function of dinB1 and dinB2 in M. tuberculosis. Mutants
with altered complements or expression levels of dinB1 and/or
dinB2 were analyzed in vitro and in vivo under conditions
predicted to be phenotypically revealing based on DinB func-
tion established with other model organisms. The lack of dis-
cernible phenotypes in any of the assays employed suggests
that the DinB homologs from M. tuberculosis do not behave
like their counterparts from other organisms.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The strains, plasmids, and oligonu-
cleotides used in this study are described in Tables S1 and S2 in the supplemental
material. M. tuberculosis strains were grown in Middlebrook 7H9 media (Difco)
supplemented with 0.2% glycerol, Middlebrook oleic acid-albumin-dextrose-
catalase (OADC) enrichment (Merck), and 0.05% Tween 80 or on Middlebrook
7H10 media supplemented with 0.5% glycerol and Middlebrook OADC enrich-
ment. Mycobacterium smegmatis strains were grown in 7H9 media supplemented
with 0.2% glycerol, 0.085% NaCl, 0.2% glucose, and 0.05% Tween 80 or on 7H10
media containing 0.5% glycerol, 0.085% NaCl, and 0.2% glucose. Hygromycin
(Hyg) and kanamycin (Km) were used at final concentrations of 50 and 25 ?g/ml,
and histidine was used at 100 ?g/ml.
Yeast two-hybrid analysis. Interaction with the replicative processivity factor
(the dnaN-encoded ?-clamp) was assessed using the Clontech Matchmaker yeast
two-hybrid (Y2H) system. The open reading frames for dinB1, dinB2, dnaE1,
dnaE2, and dnaN were PCR amplified and cloned in pGADT7 and pGBKT7 to
produce GAL4 DNA activation domain (AD) and binding domain (BD) fusions,
respectively (see Table S2 in the supplemental material), which were used in
interaction assays according to the manufacturer’s instructions.
Construction of mutant strains. The dinB1::hyg and ?dinB2 mutants of M.
tuberculosis H37Rv were prepared by two-step allelic exchange (47) using the
suicide plasmids pDINB1KO and pDINB2KO (see Table S1 in the supplemental
material). The dinB1::hyg ?dinB2 double mutant was generated from dinB1::hyg
using pDINB2KO. Genetically complemented derivatives of the single mutants
were constructed by integration of pMDINB1 and pMDINB2 at the attB site in
the dinB1::hyg and ?dinB2 mutant strains, respectively. To construct a suicide
vector for introducing a frameshift mutation at codon 205 in M. smegmatis hisD,
a 1,132-bp PstI fragment was amplified using primers HISF and HISR and
cloned in pGEM3Z(?)f. The frameshift mutation was introduced by the Mega-
primer method (57) using the mutagenic oligonucleotide HISM to form
pGHISD5T, which was used to produce pHISD5T (see Tables S1 and S2 in the
Gene expression analysis. Bacteria were grown in 7H9 media, and aliquots
sampled during different phases of growth. RNA was isolated as described
previously (16, 42), and real-time, quantitative reverse transcription-PCR (qRT-
PCR) carried out with the primer pairs detailed in Table S2 in the supplemental
material, using the LightCycler FastStart DNA Master SYBR green I kit in the
Roche LightCycler and standard curves based on 10-fold serial dilutions of
genomic DNA (29). Primers for detection of dinB1 transcript flanked the site
used for insertional inactivation with the hyg marker. Amplification conditions
were thus adjusted to ensure that only wild-type dinB1 transcript was detected.
Absolute numbers of transcripts were normalized to the number of sigA tran-
scripts in the same sample.
Sensitivity testing of M. tuberculosis strains. MICs were determined by broth
microdilution (14). Sensitivity testing was also performed by spotting 5- or 10-?l
aliquots of 3- or 10-fold serial dilutions, respectively, of parallel cultures of the
wild-type and mutant strains, grown to an optical density at 600 nm (OD600) of
?0.5 to 0.7, on Middlebrook 7H10 agar plates supplemented with compound and
scoring growth after 2 or 3 weeks.
Mutagenesis assays. Rates of spontaneous mutation of M. tuberculosis to
rifampin (Rif) resistance were determined by Luria-Delbru ¨ck fluctuation analy-
sis (48, 53) using a modification of the method described for use with M.
smegmatis (36). For each strain, 36 3-ml cultures containing an initial inoculum
of ?1 or 2 ? 103CFU/ml were set up in 6-well tissue culture plates (Greiner
Bio-One) which were incubated in a humidified incubator at 37°C for 3 weeks
with daily agitation. Aliquots from 6 cultures were serially diluted and plated on
solid media to enumerate the total colony count (nt). Each of the remaining 30
cultures was spread in its entirety onto a 7H10 agar plate containing Rif at 2
?g/ml. For analysis of mutational spectra, a representative Rif-resistant colony
was picked from each culture and a 449-bp fragment from the rpoB gene,
spanning the rifampin resistance-determining region (RRDR), was amplified
using the primers RpoBF2 and RpoBR2, and the RRDR was then sequenced
using an internal primer, RpoBS (see Table S2 in the supplemental material).
The rates of spontaneous mutation of wild-type M. smegmatis to Rif resistance or
reversion of the hisD auxotrophic mutant were determined by fluctuation anal-
ysis (36). For reversion assays, 2-ml cultures of the auxotrophic mutant HISD5T,
carrying pOLYGaa, pGaaDinB1, or pGaaDinB2 seeded at 102 to103CFU/ml,
were grown in media supplemented with histidine and Hyg to stationary phase (5
to 7 days) before plating on histidine-free 7H10 agar to determine the number of
revertants per culture. ntvalues were determined from three parallel cultures
plated on histidine-supplemented media, and mutation rates determined as pre-
viously described (36).
M. tuberculosis growth in macrophages. Human peripheral blood-derived
monocytes (PBMCs) and peritoneal macrophages from 10- to 12-week old fe-
male B6D2F1 mice were isolated as described previously (18, 37). Infections
were carried out using a multiplicity of infection (MOI) of 1 bacillus per 2
monocytes, and bacterial CFU assessed daily for 4 days, as described previously
(37, 49). Where indicated, human PBMCs and murine macrophages were acti-
vated by the addition of lipopolysaccharide (LPS) or gamma interferon (IFN-?)
to a final concentration of 1 ?g/ml or 100 U/ml, respectively, 2 h after infection.
Aerosol infection of mice. Eight- to 10-week-old female B6D2F1 mice from
Charles River Laboratories (Wilmington, MA) were infected with M. tuberculosis
strains through the respiratory route (38). Approximately 200 to 1,000 organisms
were implanted in the lungs of each mouse, as confirmed by plating lung ho-
mogenates 3 h after infection. Bacterial loads (numbers of CFU) in lungs and
spleens of infected mice were assessed over a period of 77 to 350 days.
Statistics. The independent Student t test or paired t test was applied to
compare means from two different groups. A value of P of ?0.05 was considered
dinB1 and dinB2 are expressed in M. tuberculosis. Transcript
levels of dinB1 and dinB2 were determined by qRT-PCR
during logarithmic- and stationary-phase growth of M. tu-
berculosis in vitro (Table 1). For comparison, genes encoding
the replicative polymerase DnaE1 and the SOS-inducible
polymerase DnaE2 were included in the analysis. Transcript
was detected for all genes assessed, albeit at levels that
differed significantly between genes and as a function of
growth phase. Expression of dnaE1 was highest in early
logarithmic phase and declined markedly in stationary
phase, whereas dinB1 and dinB2 showed little change be-
tween phases. Notably, dinB2 was expressed at a level 12- to
40-fold higher than that of dinB1 (Table 1).
VOL. 192, 2010DinB HOMOLOGS OF M. TUBERCULOSIS 2221
DinB1 but not DinB2 interacts with the mycobacterial
?-clamp. DNA polymerases that bind the ?-clamp associate
with this protein via a short pentapeptide motif with the con-
sensus sequence QL[SD]LF (11). In other organisms, interac-
tions of Y family polymerases with the ?-clamp play a central
role in regulating their activity and determining access to the
DNA (1–3, 8, 17, 22, 28, 35, 62, 63, 65), suggesting that an
analogous situation may apply in mycobacteria. A consensus
?-binding motif is readily identified in M. tuberculosis DinB1
(QESLF), but not DinB2, which lacks the C-terminal region in
which this motif is located in other homologs (see Fig. S1 in the
supplemental material). To confirm the assignment, the ?-
binding potential of DinB1 and DinB2 was assessed in Y2H
analyses measuring interaction by growth on selective media
(Fig. 1A) and LacZ activity (Fig. 1B). As controls, we tested
the ability of DnaN to interact with itself; with the replicative
polymerase DnaE1, which contains a consensus ?-binding mo-
tif; or with DnaE2, which does not (6). A strong DnaN-DnaN
interaction was observed, in accordance with the homodimeric
structure of the ?-clamp. The DnaN-polymerase interactions
were consistent with bioinformatics-based predictions: whereas
DnaE1 and DinB1 interacted with DnaN, DnaE2 and DinB2
did not (Fig. 1A and B). The ability of DinB1 and DinB2 to
interact with themselves, with each other, and with DnaE1 or
DnaE2 was also assessed, but no positive interactions were
identified (data not shown).
Effect of DinB homolog deficiency on susceptibility of M.
tuberculosis to genotoxic agents and novobiocin. Recent work
has uncovered a role for DinB in error-free TLS across deoxy-
guanine lesions carrying adducts at the N2position that are
generated by treatment with agents such as benzo[a]pyrene,
acrolein, nitrofurazone (NFZ), or 4-nitroquinoline-1-oxide (4-
TABLE 1. Normalized levels of polymerase gene transcripts in M.
tuberculosis H37Rv during logarithmic- and stationary-phase
growth in liquid culture
Expression level of transcript relative to sigA (?103)a
dnaE1 dnaE2dinB1 dinB2
4.0 ? 1.3
1.3 ? 0.6
0.8 ? 0.2
0.29 ? 0.14
0.04 ? 0.01
0.01 ? 0.007
0.8 ? 0.4
0.2 ? 0.2
0.3 ? 0.1
9.8 ? 1.7
8.9 ? 1.2
7.9 ? 0.6
aAccording to this representation, a value of 10 corresponds to 1% of the level
observed for sigA.
FIG. 1. Y2H analysis of polymerase interactions with the ?-clamp. (A) Growth on selective media. Potential ?-interacting partners,
cloned as GAL4 BD fusions, were tested for interaction with AD-DnaN by cotransformation and scoring for growth on selection (LT),
medium stringency (LTH), and high stringency (LTHA) media. Individual colonies of each strain were resuspended in sterile water, and the
cell density was adjusted to an OD600of 1. Tenfold serial dilutions were then plated on media of differing stringencies. LT, Leu-Trp; LTH,
Leu-Trp-His; LTHA, Leu-Trp-His-Ade dropout-supplemented media. Screening for autoactivation of the HISD reporter was carried out by
plating cotransformants on dropout-supplemented media containing 3-amino-1,2,4-triazole (3-AT) at 0.5 to 25 mM. A 3-AT concentration
of 1 mM was found to inhibit all background growth, and all further interactions were assessed at this concentration. (B) LacZ activity.
Positive interactions were confirmed by cotransformation of the corresponding vectors into strain Y187, and LacZ assays were carried out
according to the manufacturer’s instructions. ?, P ? 0.01; ??, P ? 0.005. ? and ? denote growth or lack of growth of AH109 recombinants
on LTH dropout-supplemented media.
2222KANA ET AL. J. BACTERIOL.
NQO) (27, 34, 40, 43, 54, 56, 67). Loss of dinB also sensitized
E. coli to the cytotoxic effects of alkylating agents (4). To
determine whether the M. tuberculosis dinB homologs may
function in tolerance of damage induced by such agents, we
constructed derivatives of M. tuberculosis H37Rv carrying mu-
tant dinB1 and/or dinB2 alleles (see Fig. S2A in the supple-
mental material). The dinB1::hyg and ?dinB2 single mutants
were genetically complemented by integration, at the attB site,
of a copy of the corresponding wild-type gene expressed under
the control of its own promoter. RT-PCR analysis confirmed
that the mutations eliminated the dinB1 and dinB2 transcript
detectable in the wild type and that expression was restored in
the complemented strains (see Fig. S2B).
The mutant strains grew normally when cultured in liquid
media (data not shown). They were tested for sensitivity to
various agents in liquid culture by MIC determination. Similar
MICs were observed for NFZ (75 to 150 ?M) and 4-NQO (18
to 37 ?M) for all strains tested. Similarly, no differences in
susceptibility to the alkylating agents, ethyl methane sulfonate
(EMS) and methyl methane sulfonate (MMS) were observed,
with all strains showing MICs of 10 mM and 5 mM, respec-
tively, for these compounds. The strains were also equally
susceptible to mitomycin C (MTC; MIC of 0.07 to 0.15 ?M).
Since treatment of M. tuberculosis with novobiocin (NVB) re-
sults in induction of dinB2 expression (5), we also assessed the
effect of loss of dinB1 and/or dinB2 function on sensitivity to
this drug, but no differences were observed, with all strains
showing an MIC for NVB of 0.5 to 1 ?M.
Susceptibility was further assessed by spotting serial dilu-
tions of cultures of wild-type and mutant strains sampled at the
same stage of growth (OD600value and number of CFU/ml) on
plates containing the compounds (Fig. 2). In this assay, all
strains showed equivalent susceptibility to EMS, MMS, and
MTC. The dinB1::hyg mutant showed a slight increase in NFZ
sensitivity; however, this effect was not observed in the double
dinB1::hyg ?dinB2 mutant (Fig. 2A) and thus may not be sig-
nificant in the dinB1::hyg mutant. A slight increase in sensitivity
to 4-NQO was also observed for the ?dinB2 and ?dinB1::hyg
?dinB2 mutants in some assays. However, this defect was not
reversed in the ?dinB2 attB::dinB2 mutant in which dinB2
expression was restored (Fig. 2B) and therefore is unlikely to
be associated with the loss of dinB2 function. Although it grew
as well as the other strains in liquid culture, as determined by
the OD600, the complemented dinB1 mutant surprisingly dis-
played poorer growth on solid media than either its parental
dinB1::hyg or wild-type strains (Fig. 2B). The possibility that
the growth defect of the dinB1::hyg attB::dinB1 mutant strain
was due to aberrant expression of the complementing dinB1
gene was investigated by qRT-PCR analysis. As expected, no
dinB1 transcript was detected with the dinB1::hyg mutant.
However, transcript was detected with the complemented de-
rivative at levels comparable to those of the wild type (2.3 ?
0.3% versus 2.1 ? 1.1% of the level of expression of sigA in
H37Rv and the dinB1::hyg attB::dinB1 mutant, respectively,
P ? 0.7), suggesting that the growth defect of the dinB1::hyg
attB::dinB1 mutant on solid media was not due to aberrant
expression of dinB1 integrated at the attB locus.
Role of DinB homologs in mutagenesis. The contribution of
the DinB homologs to the fidelity of replication in M. tubercu-
losis was evaluated by determining the rates of spontaneous
mutation to Rif resistance in the mutant strains. All strains
showed similar mutation rates (2.3 ? 10?9mutations per cell
per generation for the dinB1::hyg, ?dinB2, and ?dinB1::hyg
?dinB2 mutants versus 2.9 ? 10?9for M. tuberculosis H37Rv).
FIG. 2. Effect of DinB1 and/or DinB2 deficiency on susceptibility of M. tuberculosis to genotoxic agents and novobiocin. Ten or five microliters
of neat culture (?106CFU) and 10-fold (A) or 3-fold (B) serial dilutions thereof were spotted on plates containing compounds at the indicated
concentrations. Plates were incubated for 2 or 3 weeks before scoring growth.
VOL. 192, 2010 DinB HOMOLOGS OF M. TUBERCULOSIS2223
The spectrum of rpoB mutations was assessed by sequencing
the RRDR in 23 or 24 Rif-resistant mutants isolated from
different parallel cultures of each strain (Table 2). The spec-
trum was consistent with that reported for in vitro-selected
Rif-resistant mutants of M. tuberculosis H37Rv (41), and the
distributions were similar between strains. In all strains, the
Ser5313Leu and His5263Arg mutations, which are the result
of C3T transitions, were most common. The His5263Arg
mutation, resulting from an A3G transition, was also ob-
served with all strains, albeit at a slightly lower frequency in the
dinB1::hyg mutant. In addition to these dominant mutations,
C3G and A3C transversion mutations conferring a Rif-re-
sistant phenotype were also observed with some strains.
Elevated expression of dinB homologs has been shown to
confer a mutator phenotype in other organisms (31, 54). We
therefore investigated the effects of ectopic expression of M.
tuberculosis dinB1 or dinB2 on mutagenesis in the nonpatho-
genic saprophyte M. smegmatis. The M. tuberculosis genes were
cloned on an episomal vector under the control of the M.
smegmatis acetamidase promoter (46). RT-PCR analysis con-
firmed expression of both genes, even in the absence of acet-
amide (see Fig. S3 in the supplemental material), allowing
mutation rates to be assessed in the absence of inducer. How-
ever, ectopic expression of M. tuberculosis dinB1 or dinB2 had
no effect on the rate of mutation of M. smegmatis to Rif
resistance (Table 3). Since the rpoB reporter is restricted in
terms of the spectrum of mutations it can detect (base substi-
tutions), we developed a novel reporter for assessing frame-
shift mutagenesis at a homopolymeric run in mycobacteria by
transferring a ?1 chromosomal frameshift mutation into a 4T
run located in the hisD gene of M. smegmatis (23). The muta-
tion resulted in histidine auxotrophy through abrogation of the
hisD function (E. E. Machowski, unpublished data). The M.
tuberculosis dinB1 and dinB2 expression vectors were intro-
duced into the auxotroph HIS5T, and the rates of reversion to
histidine prototrophy by ?1 frameshift mutagenesis (5T34T)
compared. However, no significant differences were observed
between strains (Table 3).
dinB1 and dinB2 are individually dispensable for growth of
M. tuberculosis in macrophages and mice. To investigate the
role of the DinB homologs in virulence, intracellular growth
was assessed in various macrophage infection models. No sig-
nificant growth differences were observed between the wild
type and the dinB1::hyg mutant in resting human PBMCs or in
murine peritoneal macrophages or bone marrow-derived mac-
rophages (see Fig. S4A in the supplemental material and data
not shown). Moreover, growth of the dinB1::hyg and ?dinB2
mutants in LPS-activated human PBMCs was not significantly
different from that of the wild type (see Fig. S4B and C).
Groups of B6D2F1 mice were then aerosol infected with the
single mutant or wild-type strains, and bacillary growth was
monitored over time. The dinB1::hyg mutant, seeded at 2.5 ?
0.2 log10CFU, displayed no attenuation of growth in the lungs
of infected mice compared to the wild-type strain, seeded at
2.3 ? 0.2 log10CFU. The lung bacillary loads of the mutant
and wild-type strains were comparable; the 1.2 log10-higher
number of CFU of the mutant over the wild type at 350 days
postinfection was not statistically significant (P ? 0.08) (Fig.
3A). The mutant was also fully proficient in hematogenous
dissemination to the spleen, and no significant differences in
survival of mice infected with the dinB1::hyg mutant were
noted (data not shown). The ?dinB2 mutant, inoculated at
2.9 ? 0.3 log10CFU, displayed slightly reduced bacillary loads
in the lung compared to the wild type (seeded at 3.2 ? 0.3 log10
CFU) during early infection but achieved comparable numbers
of CFU during late infection (P ? 0.1) (Fig. 3B). Similarly,
dissemination of this mutant to the spleen was reduced during
early infection, possibly due to the reduced lung bacillary
loads, but this effect was lost over time with no difference in the
number of spleen CFU between the wild type and the ?dinB2
mutant recorded at 315 days postinfection (data not shown).
To further analyze the virulence of the ?dinB2 mutant during
early infection, bacillary loads in the lungs of mice infected
with the ?dinB2 mutant and wild-type strains were assessed
alongside the complemented mutant in a separate experiment.
However, no significant differences were observed (Fig. 3B,
TABLE 2. Spectrum of rpoB mutations in spontaneous Rif-resistant mutants derived from M. tuberculosis mutant strains
lacking dinB1 and/or dinB2
Frequency of mutation (%)a
aTwenty-three independent Rif-resistant mutants were analyzed for H37Rv and the dinB1::hyg and ?dinB2 mutants, and 24 mutants were analyzed for the
?dinB1::hyg ?dinB2 mutant.
TABLE 3. Mutation rates for M. smegmatis strains overexpressing
M. tuberculosis dinB1 and dinB2
Rif resistance (rpoB)
aNA, not applicable.
2224 KANA ET AL.J. BACTERIOL.
Insight into the expression, activity, and regulation of spe-
cialized polymerases belonging to the DinB subfamily of Y
family polymerases has been gained from studies of E. coli
(reviewed in reference 26), P. aeruginosa (54), and Bacillus
subtilis (17). Important similarities and differences have been
identified between organisms in terms of the number of DinB
homologs and the complement of other DNA polymerases that
are present, the mechanisms regulating their expression (SOS
regulated or not), interaction with the ?-clamp and the func-
tional significance thereof, and the effects of loss-of-function
mutations or DinB overproduction on susceptibility to DNA
damage and/or mutagenesis. Therefore, defining a “canonical”
role for DinB in bacterial DNA metabolism has proven to be
elusive, although some characteristic functions have emerged,
such as error-free TLS across N2-dG lesions.
In this study, we investigated the role of the two DinB
homologs in M. tuberculosis. We identified marked differences
between DinB1 and DinB2 at the level of expression and
interaction with the ?-clamp and also between them and ho-
mologs in other bacteria, in terms of protection against the
cytotoxic effects of DNA damage and mutator activity. Se-
quence analysis suggests that DinB1 and DinB2 may be func-
tional DNA polymerases (see Fig. S1 in the supplemental
material). Attempts were made to confirm this prediction by
overexpression, purification, and enzymatic characterization of
these proteins as E. coli recombinants. However, these efforts
met with limited success (data not shown), and as a result, the
DNA polymerase activities of DinB1 and DinB2 have yet to be
The level of sigA-normalized dinB2 transcript detected in
cultures of M. tuberculosis grown in vitro was significantly
higher than that of dinB1 and also higher than that of dnaE1,
which encodes the replicative polymerase. Moreover, unlike
dnaE1, which was expressed at lower levels in stationary- ver-
sus logarithmic-phase cells, the levels of dinB1 and dinB2 tran-
script did not change significantly as a function of growth
phase. The comparatively high level of dinB2 expression in M.
tuberculosis is therefore reminiscent of the situation in E. coli
under non-SOS-inducing conditions, in which DinB is present
at a level more than 10-fold higher than that of the Pol III
One feature of M. tuberculosis DinB2 that differentiates it
from DinB1 and the DinB homologs from E. coli and B. subtilis
is an inability to bind to the ?-clamp, which may impair or even
preclude putative TLS and replication slow-down functions
(10, 25). Thus, overexpression of dinB2 would not be expected
to affect the growth or colony-forming ability of M. tuberculosis,
whereas overexpression of dinB1, which encodes a protein with
?-binding activity, might. However, aberrant dinB1 expression
was ruled out as an explanation for the impaired colony-form-
ing ability of the dinB1::hyg attB::dinB1 mutant, a phenotype
which has yet to be resolved. Therefore, the functional impli-
cations of the differential ?-binding activity of DinB1 and
DinB2 remain unclear and await further information on their
DNA polymerase activity, the importance (or otherwise) of
?-binding for polymerase function in mycobacteria and asso-
ciation between ?-binding by a specialized DNA polymerase,
replication fork slowdown, and the effects thereof on mycobac-
terial growth (25, 61).
The loss of dinB1 and/or dinB2 function had little or no
effect on the sensitivity of M. tuberculosis to N2-dG adduct-
forming compounds (27, 54) or to alkylating agents (4), in
contrast to that of other organisms. While evidence suggests
that the in vivo environments encountered by M. tuberculosis
are genotoxic, the types of DNA lesions generated in these
environments have not been defined (6, 13, 55). The lesion
N2-(1-carboxyethyl)-2?-dG (N2-CEdG) has been detected with
human melanoma cells, and its frequency shown to increase in
a dose-responsive manner by treatment with methylglyoxal
(MG) or glucose (67). Interestingly, transcriptional profiling of
M. tuberculosis in pulmonary TB has shown that the tissue
environment of M. tuberculosis at sites of infection is rich in
MG (51), which is present at elevated levels in macrophages
during mycobacterial infection (50). The upregulation of dinB1
observed during pulmonary TB (51) suggests a plausible link
between the production of host and/or M. tuberculosis-derived
MG during infection, the induction of N2-CEdG lesions in M.
tuberculosis, and dinB1 function. However, the dispensability of
DinB1 (and DinB2) for the growth of M. tuberculosis in vivo
FIG. 3. Growth and survival of DinB1 and DinB2 mutants of M. tuberculosis H37Rv in infected mice. Growth of the dinB1::hyg (A) and ?dinB2
(B) mutants with the parental wild type was monitored over a period of 350 and 315 days postinfection, respectively. The inset in panel B shows
growth and survival of the ?dinB2 mutant, its complemented derivative, and the parental wild type over a period of 77 days postinfection. Three
mice were sacrificed at each time point for each strain. ?, wild type; ?, dinB1::hyg mutant; ‚, ?dinB2 mutant; E, ?dinB2 attB::dinB2 mutant.
VOL. 192, 2010DinB HOMOLOGS OF M. TUBERCULOSIS2225
suggests that M. tuberculosis possesses another mechanism(s)
for dealing with the cytotoxic effects of lesions, such as N2-
CEdG, that may obscure the effects of DinB homolog defi-
ciency. One possibility is nucleotide excision repair (NER),
which provides the major mechanism for processing NFZ-
induced damage in E. coli (45). NER has been implicated in
the virulence of M. tuberculosis in mice (13) through the pro-
cessing of DNA damage caused by nitrosative stress (12).
Given the comparatively low rate of DNA replication in M.
tuberculosis (24), NER may play an even more dominant role
than damage tolerance in enabling this organism to contend
with such lesions.
Consistent with the findings for E. coli (33), the loss of dinB1
and/or dinB2 function had no effect on the replication fidelity
of M. tuberculosis in vitro, as evidenced by the rate of sponta-
neous mutation to Rif resistance. Minor differences in the
spectrum of rpoB mutations were observed between strains,
but in all cases, Rif resistance arose predominantly from C3T
mutations. Instead, our results suggest the value of developing
a mutational reporter suitable for readout of the G3T muta-
tions that might be expected to increase in frequency in strains
defective in error-free bypass of lesions such as N2-CEdG (67).
However, in a further deviation from findings for other organ-
isms (31, 54), ectopic expression of the M. tuberculosis dinB
homologs had no discernible effect on rates of (base substitu-
tion) mutation to Rif resistance or ?1 frameshift mutagenesis
at a homopolymeric (5T) run in M. smegmatis. Again, it is
possible that the reporters employed are not suitable for de-
tecting mutations that the DinB homologs may be particularly
prone to inducing, such as ?1 frameshifts within homopoly-
meric runs of deoxyguanylic acids (31, 54) or alternatively that
the M. tuberculosis dinB1 and dinB2 expression levels were not
sufficiently high to confer an effect. To test these possibilities,
a broader range of mutational reporters and further expression
vectors are under development in our laboratory.
In conclusion, the physiological roles of DinB1 and DinB2 in
DNA metabolism in M. tuberculosis have proven remarkably
refractory to elucidation using models that were predicted to
be phenotypically revealing on the basis of insights from other
organisms. Together, our findings suggest that the DinB ho-
mologs from M. tuberculosis differ significantly from their
counterparts from other bacteria.
This work was funded by grants from the Wellcome Trust (Collab-
orative Research Initiative grant 065578 to N.G.S. and V.M.), the
Medical Research Council of South Africa (to V. M.), the National
Research Foundation (to V. M.), the National Institutes of Health
(R01 AI 54338 to G.K.), the Howard Hughes Medical Institute (In-
ternational Research Scholar’s grant to V.M.), and the Welch Foun-
dation (to J.C.S.). B.D.K. was supported by a traineeship from the
Columbia University-Southern Africa Fogarty AIDS International Re-
search and Training Program (NIH/FIC D43 TW00231 [to Q. Abdool
Karim]). N.S. was supported by a postdoctoral fellowship from the
Heiser Program for Research in Leprosy and Tuberculosis.
We thank Stewart Cole for providing BACC12, Tanya Parish for
providing pAGAN11, Dorothy Fallows for constructively reviewing the
manuscript, members of the Mizrahi, Kaplan, and Sacchettini labora-
tories for advice and assistance, and anonymous reviewers for helpful
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