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The Mycobacterium tuberculosis IdeR is a dual

functional regulator that controls transcription of genes
involved in iron acquisition, iron storage and survival in
Benjamin Gold,
G. Marcela Rodriguez,
Salvatore A.
E. Marras,
Mickey Pentecost
and Issar Smith
Department of Microbiology, New York University
Medical Center, New York, NY 10016, USA.
TB Center, Public Health Research Institute, 455 First
Avenue, New York, NY 10016, USA.
Albert Nerkin School of Engineering, Cooper Union, New
York, NY, USA.
In this work, we characterize genes in Mycobacterium
tuberculosis that are regulated by IdeR (iron-depen-
dent regulator), an iron-responsive DNA-binding pro-
tein of the DtxR family that has been shown to regulate
iron acquisition in Mycobacterium smegmatis. To
identify some of the genes that constitute the IdeR
regulon, we searched the M. tuberculosis genome for
promoter regions containing the consensus IdeR/DxR
binding sequence. Genes preceded by IdeR boxes
included a set encoding proteins necessary for iron
acquisition, such as the biosynthesis of siderophores
(mbtA, mbtB, mbtI ), aromatic amino acids ( pheA, hisE,
hisB-like) and others annotated to be involved in the
synthesis of iron-storage proteins (bfrA, bfrB). Some
putative IdeR-regulated genes identified in this search
encoded proteins predicted to be engaged in the
biosynthesis of lipopolysaccharide (LPS)-like mol-
ecules (rv3402c), lipids (acpP) and peptidoglycan
(murB). We analysed four promoter regions containing
putative IdeR boxes, mbtA–mbtB, mbI, rv3402c and
bfrA–bfd, for interaction with IdeR and for iron-
dependent expression. Gel retardation experiments
and DNase footprinting analyses with purified IdeR
showed that IdeR binds to these IdeR boxes in vitro.
Analysis of the promoters by primer extension
indicated that the IdeR boxes are located near the
210 position of each promoter, suggesting that IdeR
acts as a transcriptional repressor by blocking RNA
polymerase binding. Using quantitative reverse tran-
scriptase–polymerase chain reaction (RT–PCR)
coupled to molecular beacons, we showed that
mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd
are induced 14- to 49-fold in cultures of M.
tuberculosis starved for iron, whereas mRNA levels
of bfrA decreased about threefold. We present
evidence that IdeR not only acts as a transcriptional
repressor but also functions as an activator of bfrA.
Three of the IdeR- and iron-repressed genes, mbtB,
mbtI and rv3402c, were induced during M. tubercu-
losis infection of human THP-1 macrophages.
The low levels of available iron in a bacterial pathogen’s
mammalian host (estimated at 10
M; Neilands, 1995)
may potentiate the expression of genes encoding iron
acquisition systems, virulence factors and toxins (Muller
et al., 1983). Iron limitation by the human body is
exacerbated by an upregulation of the expression of
transferrin, lactoferrin, ferritin and other iron-sequestering
molecules in response to bacterial infections (Leffel and
Spitznagel, 1975; Wright and Gallin, 1979; Muller et al.,
1983; Weinberg, 1984). These molecules have a high
affinity for iron and sequester available free iron required
for bacterial growth. Therefore, many bacterial pathogens,
such as Yersinia, Salmonella and Legionella, require iron
acquisition systems to survive the iron-limited environment
of their host (Pope et al., 1996; Bearden and Perry, 1999;
Janakiraman and Slauch, 2000). Recent results suggest
an essential role for iron acquisition in Mycobacterium
tuberculosis, the aetiological agent of tuberculosis, during
infection. A null mutation of mbtB in M. tuberculosis,
encoding an enzyme involved in the biosynthesis of the
siderophore mycobactin (Quadri et al., 1998), was shown
to have impaired growth in the human macrophage cell
line THP-1 (De Voss et al., 2000). Furthermore, mbtB
expression is induced up to 60-fold during M. tuberculosis
infection of mice (J. Timm et al., unpublished). These data
imply a crucial role for mycobactin-mediated iron
acquisition for M. tuberculosis virulence in both macro-
phage and murine models. If M. tuberculosis faces
low-iron conditions in a host, we can hypothesize that
Accepted 29 August, 2001. *For correspondence at the TB Center.
E-mail; Tel. (11) 212 578 0868; Fax (11) 212
578 0804.
Molecular Microbiology (2001) 42(3), 851–865
Q 2001 Blackwell Science Ltd
many iron-regulated genes will be upregulated in the course
of an infection, and that these genes could be important for
the tubercle bacillus’ adaptation and survival in the host.
Bacterial iron acquisition has been shown to be regulated
by the iron-responsive proteins of the Fur and DtxRfamilies.
IdeR (iron-dependent repressor), found in all mycobacteria
(Doukhan et al., 1995), is the structural and functional
homologue of the Corynebacterium diphtheriae DtxR
(Schmitt et al., 1995) and has been shown in Mycobacterium
smegmatis to negatively regulate siderophore biosynthesis
(Dussurget et al., 1996). A functional IdeR binding site (an
IdeR box) was found upstream of fxbA, and three other
putative IdeR binding sites were identified in the M.
smegmatis exochelin biosynthesis locus (Fiss et al., 1994;
Yu et al., 1998; Dussurget et al., 1999). The M. smegmatis
ideR mutant cannot mount an effective oxidative stress
response (Dussurget et al., 1996), probably because of
decreased expression of katG and sodA as the levels of
both mRNA and protein decline (Dussurget et al., 1996;
1998). These results suggest that IdeR is a functional
homologue of Fur, a pleiotropic regulator controlling over
60 genes in Escherichia coli involved in diverse functions
Table 1. Putative IdeR binding sites located in the M. tuberculosis H37Rv genome.
The putative IdeR box sequences in this table were identified by probing the M. tuberculosis genome with a consensus IdeR/DtxR binding sequence
using the program TUBERCULIST. Genes that have been grouped together within a black box indicate those that are divergently transcribed from the
same IdeR box. The IdeR binding sites of fxbA, from M. smegmatis, and hisE–irg2, from M. tuberculosis, are the only IdeR targets that have been
biochemically and physiologically verified to date (Dussurget et al., 1999; Rodriguez et al., 1999).
a. Mis., mismatches from the consensus sequence.
b. Rv#: indicates the numerical designation of each open reading frame in the annotated genome. The ‘c’ that follows some Rv#s indicates that the
gene has a reverse orientation. An asterisk (*) indicates IdeR boxes that have been validated.
c. Bases with a black background match the query sequence. The letter W represents weak basepairing (the bases A or T), and S is for strong
basepairing (G or C).
d. Location indicates the distance of the 5
end of the IdeR box to the predicted ORF start site.
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ranging across iron homeostasis, oxidative stress, toxin
synthesis and general metabolism (Hantke, 2001).
However, the entire IdeR regulon has yet to be defined,
nor have any direct positive targets been identified.
In this work, we have identified IdeR-regulated genes
in M. tuberculosis using a computer-based approach to
search the recently published genome sequence (Cole
et al., 1998). Four promoter regions containing putative
IdeR boxes were shown to bind purified IdeR, and
DNase protection analysis showed that IdeR protects the
predicted IdeR box sequence. The transcription of the
six genes downstream of these IdeR boxes (mbtA,
mbtB, mbtI, rv3402c, bfd and bfrA) was induced by
growth in an iron-limited medium. In addition to IdeR’s
role as a repressor, we provide evidence that IdeR
activates bfrA transcription directly. In addition, three
IdeR-repressed genes were found to be induced during
growth of M. tuberculosis in human THP-1
Identification of IdeR boxes
IdeR was predicted to bind a similar DNA sequence to the
C. diphtheriae DtxR, given the following observations: the
DNA-binding domain of IdeR is 92% identical and 100%
similar on the amino acid level with that of DtxR (Doukhan
et al., 1995); these proteins have almost identical crystal
structures (Pohl et al., 1999); IdeRcan complement a dtxR
mutant for iron-dependent regulation of DtxR-regulated
promoters (Schmitt et al., 1995); and finally, IdeR binds to
the C. diphtheriae tox promoter and other DtxRtarget gene
promoters (Schmitt et al., 1995). Therefore, we hypoth-
esized that many of the IdeR boxes in the M. tuberculosis
genome could be identified by conducting a computer
search using a DtxR binding site sequence as a surrogate.
A preliminary search for IdeR boxes using the BLAST
program against every published sequence (not specific
for M. tuberculosis ) resulted in the identification of the M.
tuberculosis hisE–irg2 promoter region (Rodriguez et al.,
Fig. 1. IdeR binds DNA fragments containing the predicted IdeR box. IdeR was added in fourfold increasing concentrations, from 0.06 to 1 mM, to
P-labelled DNA probes in the presence of 200 mM Ni
, and complexes were resolved on an 8% Tris acetate polyacrylamide gel containing
200 mM Ni
. The probe alone without added protein is in the left-most lane and indicated by (–). Promoter regions containing one iron box included
mbtA–mbtB (A), mbtI (B) and rv3402c (C), whereas bfrA–bfd (D) contains two iron boxes. Control gel retardation experiments using DNA fragments
without an IdeR box showed no interaction with the IdeR protein (data not shown). U, unbound probe in the absence of IdeR; B1, B2 and B3, bound
probe in the presence of IdeR.
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1999). We have nowconducted a broader search of the M.
tuberculosis published genome (Cole et al., 1998) for IdeR
boxes to identify genes regulated by IdeR using the
available computer program TUBERCULIST (http://genolist. Moszer et al., 1995) (Table1). The
criteria chosen as qualities for a DNA region regulated by
IdeR were limited to those within 1250 to 1100bp of the
predicted start codon of an open reading frame (ORF) and
containing five or fewer mismatches from our query
sequence of TWAGGTWAGSCTWACCTWA (where
W¼A or T, and S¼G or C). There are two major reasons
why the putative IdeR boxes in Table 1 are limited to five
mismatches. First, there was a dramatic increase in
potential IdeR binding sites with each subsequent
mismatch allowed (estimated as more than 100 targets
for six mismatches and more than 700 targets for seven
mismatches). Secondly, if IdeR boxes were randomly
distributed in the M. tuberculosis chromosome, there
should be approximately four times more putative IdeR
boxes inside an average length ORF (<1000 bp)
compared with a 250bp promoter region. This was not
the case, as there was only a small number of putative
IdeR binding sites with four mismatches (two in ORFs
versus 17 located 250 bp upstream of the putative
translational start site), whereas five mismatches resulted
in 16 in ORFs versus 19 located 250bp upstream of the
putative translational start site. Although this indicated that
the computer search for IdeR boxes was probably more
accurate with #four mismatches, putative binding sites
with five mismatches were left in Table 1, as some of these
were located upstream of genes whose transcription was
predicted to be regulated by IdeR (annotated to be
involved in iron storage and acquisition).
Over 40 genes preceded by putative IdeR boxes were
Fig. 2. IdeR binds to predicted IdeR box sequences.
P-labelled fragments were bound to 1 mM purified IdeR in the presence of 200 mM Ni
subjected to DNase digestion. Maxam–Gilbert A1G sequencing reactions were performed on the same probes to identify the exact sequence to
which IdeR binds. Reaction products were separated on a 6% TBE polyacrylamide sequencing gel containing 8 M urea. Protected regions are
marked with a black line, and a summary of these sequences is shown in Fig. 3.
A. mbtA top strand.
B. mbtB top strand.
C. mbtI bottom strand.
D. mbtI top strand.
E. rv3402c bottom strand.
F. rv3402c top strand.
G. bfd top strand.
H. bfrA top strand.
A/G, Maxam–Gilbert A1G sequence ladder; –, no IdeR added, 1, 1 mM IdeR.
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identified and fell into several categories (Table 1). One
group encodes proteins that are annotated to be directly
involved in either iron acquisition (mbtA–mbtB, mbtI,
rv1348, rv1347c ) or iron storage (bfrA, bfrB). The second
group of genes, encoding annotated proteins involved in
amino acid biosynthesis (hisB, hisB-like, pheA, lysyl
tRNA) may be involved in siderophore biosynthesis. The
last group contains many genes that either have no
obvious relationship to iron (murB, acp) or have no
significant homologies to any known proteins in
To test the functionality of the IdeR boxes identified by
the computer search, we analysed four that were between
or in front of the following genes: mbtA–B, mbtI, rv3402c
and bfd–bfrA. mbtA, mbtB and mbtI encode enzymes
involved in the biosynthesis of mycobactin, the major M.
tuberculosis siderophore (Quadri et al., 1998; De Voss
et al., 2000). rv3402c encodes a protein with .40% amino
acid similarity to three types of enzymes: an aminotrans-
ferase, a dehydratase and an enzyme involved in
perosamine/O-antigen biosynthesis. bfrA encodes a
bacterioferritin subunit that is involved in iron storage.
bfd is not currently annotated in the M. tuberculosis
genome, but we found that this ORF has homology (30–
40% amino acid identity, .50% conserved amino acids)
to Bfd from Escherichia coli, a protein that associates with
BfrA and may have a role in donating iron to the
bacterioferritin complex (Garg et al., 1996; Quail et al.,
1996). In M. tuberculosis, bfd and bfrA are adjacent and
divergently transcribed (Fig. 5C) and, in E. coli, bfd and
bfrA are also neighbouring genes but are transcribed in the
same direction (Andrews et al., 1993).
IdeR binds to DNA fragments containing putative IdeR
To demonstrate that IdeR could bind to the putative IdeR
boxes identified from our computer search, DNA frag-
ments containing these sequences were PCR amplified
and labelled with
P for gel retardation analysis. The
labelled probes were mixed with various concentrations of
purified IdeR (0.06–1 mM) and then separated on 8%
polyacrylamide gels (Fig. 1). Nickel was used as the
divalent metal in the binding reactions on account of its
redox stability compared with ferrous iron. The specificity
of purified IdeR to its target sequences has been
demonstrated in a previous work (Rodriguez et al.,
1999). In addition, we have shown previously IdeR’s
broad in vitro divalent metal specificity and IdeR’s metal
dependence for function (Schmitt et al., 1995; Dussurget
et al., 1996).
The migration of all four DNA fragments tested was
retarded by IdeR in a concentration-dependent manner
(Fig. 1). The labelled probes from mbtA–B, mbtI and
rv3402c, with one IdeR box, had one major shifted product
(B2), whereas bfd–bfrA, containing two IdeRbinding sites,
had two major shifted products (B2 and B3). All four
fragments exhibited one minor intermediate band (B1) that
appeared with the lower concentrations of IdeR, which
may represent an intermediate with one IdeR dimer bound
to the probe (White et al., 1998). These results indicate
that IdeR can bind to these DNA fragments that contain
putative IdeR-binding sequences.
IdeR binds to IdeR box sequences
We performed DNase protection assays with IdeR bound
to the four DNA fragments containing putative IdeR boxes,
mbtA–B, mbtI, rv3402c and bfd–bfrA, in order to identify
the DNA sequences bound by the protein. The results of
the DNase footprinting experiments are shown in Fig. 2,
and the sequences protected by IdeR are summarized in
Fig. 3. IdeR protected the predicted IdeR box sequence for
mbtA–mbtB, mbtI, rv3402c and both IdeR boxes for bfd–
bfrA. The protection pattern covers the 19 bp IdeR box
sequence and also includes up to a 10 bp overhanging
region of protection 3
to the end of the IdeR box on both
the top and the bottom strand. The sequences protected
by IdeR are similar to those found in our previous studies
with the M. smegmatis fxbA and the M. tuberculosis hisE–
irg2 (Dussurget et al., 1999; Rodriguez et al., 1999), and
these are included here for comparison (Fig. 3).
IdeR functions as a repressor by blocking the 210 region
of the promoter
We mapped the transcriptional start points (TSP) of mbtB,
mbtI, rv3402c, bfd and bfrA by primer extension analysis
using RNA from M. tuberculosis grown in either low- or
high-iron-containing medium (Figs 5 and 6; data not
shown), and the results are summarized in Fig. 3. The
mRNA levels of mbtA were too low for this analysis. The
TSP of mbtB, mbtI, rv3402c, bfrA and bfd all mapped
within the IdeR box or 5 bp downstream of the box. We
identified putative 210 sequences with similarity to the
sequence TAgRcT (Gomez and Smith, 2000) that were
the correct distance from the TSPs (Fig. 3). The mapping
of the TSPs indicates that these IdeR boxes are located in
close proximity to the 210 region, which fits a model that,
in the presence of iron, IdeR functions as a repressor by
occluding the site of RNApolymerase binding (Fig. 8), as is
the case for DtxRbinding to its target genes (Schmitt et al.,
Genes preceded by an IdeR box are regulated by iron
The expression of genes with IdeR boxes was analysed in
M. tuberculosis cultures grown with low and high levels of
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iron by reverse transcriptase–polymerase chain reaction
(RT–PCR) coupled with molecular beacons (mbRT–
PCR) (Manganelli et al., 1999). We used the sigA mRNA
for normalization, as its levels remain constant in low- and
high-iron concentrations (data not shown; Dussurget et al.,
1999; Rodriguez et al., 1999). The average ratio of mRNA
copies in low/high iron were 14 (mbtA), 49 (mbtB), 17
(mtbI ), 17 (rv3402c ) and 40 (bfd) (Fig. 4). The effect of
iron on bfrA expression is described in the next section. As
was predicted from the presence of an IdeR box in their
promoter region, the transcription of these genes was
indeed repressed by high iron levels.
IdeR acts positively on bfrA expression
We expected that the expression of bfrA, encoding an iron
storage protein, bacterioferritin, would be upregulated in
high-iron conditions as has been shown in other bacteria
(Andrews, 1998; Miller et al., 2000). However, as all the
Fig. 3. Summary of data from DNase footprinting and transcriptional start point analysis. The predicted IdeR binding sequence is indicated in capital
letters, and the region on either strand protected fromDNase digestion by IdeRis indicated by shading with black. Transcriptional start points (TSPs)
for each gene were mapped by primer extension and verified with two separate primers, using RNA prepared from M. tuberculosis grown in low- and
high-iron medium. Data for the bfrA TSPs are shown in Fig. 5; others are not shown. The TSP has not yet been mapped for mbtA. For comparison,
data on IdeR’s interaction with the promoters of the M. smegmatis fxbA and the M. tuberculosis hisE–irg2 and their TSPs in low iron have been
included (Dussurget et al., 1999; Rodriguez et al., 1999).
Fig. 4. Expression of genes preceded by IdeR boxes during iron
limitation. RNA was prepared fromM. tuberculosis H37Rv after growth
in a minimal medium containing 50mM FeCl
or lacking iron for six
generations. Using gene-specific molecular beacons, low-/high-iron
induction ratios were calculated with quantitative molecular beacon
RT–PCR. Gene expression was normalized to sigA, whose mRNA
levels are not affected by iron concentration.
856 B. Gold et al.
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other IdeR- and DtxR-regulated genes have been shown to
berepressedbyhighironlevels(Schmitt et al., 1992; Schmitt
and Holmes, 1994; Lee et al., 1997; Dussurget et al., 1999;
Rodriguez et al., 1999), it was intriguing to study how bfrA
would be regulated by IdeR. In addition, the bfrApromoter is
unlike the other IdeR-regulated genes because it has two
tandemIdeR boxes, beginning at 207 and 231bp upstream
of the annotated ORF start site (Fig. 5).
Fig. 5. bfrA low- and high-iron promoters. RNA from a culture grown in conditions of iron limitation was isolated from both M. tuberculosis and M.
smegmatis carrying an M. tuberculosis bfrA–lacZ fusion. The 5
mRNA termini were mapped using
P-labelled reverse primers specific to the M.
tuberculosis bfrA mRNA. The TSPs were identical when using total RNA isolated from M. tuberculosis H37Rv or M. smegmatis MC
155 harbouring
the M. tuberculosis bfrA–lacZ: (A) P
using RNA from cells grown in a medium containing high iron; and (B) P
and P
, using RNA from cells
grown in an iron-limiting medium. The locations of P
, P
and P
in the bfrA upstream region are shown in (C), where the two IdeR boxes are
identified by bold lettering.
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As we speculated that bfrA might be positively regulated,
we wanted to determine whether the expression of this gene
required IdeR and the two IdeR binding sites. To address
these questions, a bfrA–lacZ fusion construct was made
that contained 378bp upstream from the bfrA initiating
codon and was integrated into the chromosome of wild-type
M. smegmatis MC
155 and its isogenic ideR mutant, SM3
(Dussurget et al., 1996). In addition, a bfrA–lacZ fusion was
made with the tandem IdeR boxes deleted (bfrADIB–lacZ),
and this construct was also incorporated into MC
155 and
Initial experiments using the intact bfrA–lacZ fusion
integrated into M. smegmatis MC
155 demonstrated that
levels of bfrA decreased (<twofold) during iron limitation.
When the bfrA–lacZ and bfrADIB–lacZ constructs were
integrated into M. smegmatis SM3, there was little or no
Fig. 6. IdeR and iron-dependent regulation of bfrA. A map of the bfrA promoter region is summarized in (A), showing the location of the two IdeR
boxes (IB) and P
, P
and P
. The expression of P
in high and low iron was analysed using M. smegmatis strains carrying bfrA–lacZ (B)
and a strain carrying a bfr –lacZ with two IdeR boxes deleted, bfrADIB–lacZ (C). P
expression in high and low iron was analysed only using the
bfrA–lacZ (F), as the deletion of the IdeR boxes abrogates P
activity. The regulation of bfrA was analysed by mbRT–PCR using a beacon that
recognizes a sequence common to the mRNAs produced by P
and P
, and recognizing a sequence 5
to P
and unique to P
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lacZ expression (data not shown). The analysis of the bfrA
promoter region was extended to locate the TSP, using
RNA isolated fromthe M. smegmatis strains containing the
M. tuberculosis bfrA–lacZ and bfrADIB–lacZ fusions. The
primers used were specific for the M. tuberculosis bfrA and
not endogenous M. smegmatis bfrA, as the primer
extension products were not seen using RNA isolated
from M. smegmatis not containing the M. tuberculosis
promoter fusions (data not shown). The TSPs of bfrA, from
both M. tuberculosis total RNA and the M. smegmatis
strain carrying the M. tuberculosis bfrA–lacZ fusion were
identical when grown in both high iron (Fig. 5A) and low
iron (Fig. 5B), and the location of these TSPs is shown in
Fig. 5C. The stronger band intensity of the M. tuberculosis
and P
in Fig. 5B results from longer iron
starvation. These results indicated that the regulation of
the M. tuberculosis bfrA could be studied using M.
smegmatis as a surrogate host.
A TSP was detected in high iron and was located 110bp
upstreamof the ATG(P
) (Figs 5A and 6B). P
is decreased during iron starvation (Fig. 6B, lanes 1 and 2)
and is dependent on IdeR, as the levels of P
are very low in the ideR mutant strain (Fig. 6B, lanes 3 and
4). If the IdeR boxes are deleted (bfrADIB–lacZ), the
expression of P
in high iron is reduced (Fig. 6C, lanes 1
and 2) compared with when the IdeR boxes are present
(Fig. 6B, lanes 1 and 2). A combination of deleting the IdeR
boxes and lacking IdeR completely abrogated bfrA P
activity (Fig. 6C, lanes 3 and 4). The mRNA produced by
is not a processed mRNA produced by P
, as it is
made, albeit at lower levels, even when P
is deleted
(Fig. 6C, lanes 1 and 2). Two primer extension products
were present only during iron starvation, which mapped to
the distal IdeR box (P
and P
) (Figs 5B and 6D).
and P
were always present in equal amounts and
will henceforth be named P
to refer to both promoters.
The repression of P
in high iron is dependent on IdeR,
as the transcription of P
remains high in the ideR mutant
(Fig. 6D). The activity of P
was estimated to be 5–10
times stronger than P
by quantitative RT–PCR and
band intensity from primer extension (data not shown).
The contributions of P
and P
were measured more
precisely using mbRT–PCR of M. tuberculosis RNA
derived from cultures grown in low- and high-iron medium.
The total amount of bfrA mRNA (P
) decreased in
a time-dependent manner to about 30% during iron
starvation (Fig. 6E). Using RT–PCR with a beacon
specific for the P
mRNA (which recognizes a sequence
to P
), P
was demonstrated to be induced about
11-fold during iron starvation (Fig. 6E). The decrease in
bfrA mRNA (P
) during iron starvation can be
attributed to a combination of mRNA differentially
expressed from both P
and P
(Fig. 8). P
expressed during growth in conditions of iron excess and
requires IdeR for expression. During conditions of iron
starvation, P
is shut off, and IdeR releases its
repression of P
, allowing for low-level expression from
IdeR-controlled genes are derepressed during M.
tuberculosis infection of human macrophage-like THP-1
The levels of mbtB, mbtI, Rv3402c and bfrA were
compared in broth cultures of M. tuberculosis and M.
tuberculosis infection of the human monocytic cell line
THP-1 that had been differentiated into macrophages
(Fig. 7). THP-1 macrophages were infected with M.
tuberculosis and, at various time points, macrophages
containing internalized M. tuberculosis were harvested,
and RNA was isolated for subsequent analysis by mbRT–
PCR. sigA mRNA was used to normalize all ratios, as it
had been determined previously that the M. tuberculosis
sigA mRNA levels remain constant during M. tuberculosis
infections of THP-1 cells (Manganelli et al., 2001;
E. Dubnau et al., submitted). The expression of genes
previously reported to be expressed during intracellular
growth in macrophages, icl, encoding isocitrate lyase
(McKinney et al., 2000), and hspX, encoding a-crystallin
(Yuan et al., 1998), was analysed, and both were found to
be strongly induced (data not shown; E. Dubnau et al.,
submitted). Two genes from the mycobactin biosynthesis
operon were each induced to essentially the same levels
at both 24 and 72 h: mbtB (<38-fold) and mbtI (<fourfold)
(Fig. 7). rv3402c was induced 11-fold by 24 h, and 25-fold
Fig. 7. IdeR-regulated genes during M. tuberculosis infection of THP-1
macrophages. THP-1 macrophages were infected at a multiplicity of
infection of <1 with M. tuberculosis H37Rv, and total RNA was
extracted at 24 and 72 h after infection. mRNA levels were determined
by mbRT–PCR, and values were normalized against sigA, whose
levels remain constant during M. tuberculosis growth in vitro, during
iron starvation and in macrophages.
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by 72 h (Fig. 7). The levels of bfrA (P
) did not
change significantly from broth-grown cultures at either
time point. To control for induction of gene expression
during the 2 h period while the macrophages were
ingesting M. tuberculosis, the levels of mRNA for all the
genes were compared with cultures incubated in RPMI or
7H9 for 2h. Only mbtB was responsive to a 2 h exposure to
RPMI and was induced threefold, and the other genes
were unaffected. Accordingly, if mbtB is normalized
against RNA from M. tuberculosis exposed to RPMI for
2 h, its induction is 11-fold (versus 38 if normalized to 7H9).
The induction of the mycobactin biosynthesis genes, mbtB
and mbtI, suggests that the mycobacterial phagosome
may be limiting for iron, consistent with previous findings
(De Voss et al., 2000).
We identified putative IdeR-regulated genes as the result
of a computer search for IdeRbox-like sequences in the M.
tuberculosis genome. Six genes selected to perform DNA-
binding experiments and quantitative analysis of gene
expression were all regulated by iron levels and IdeR. This
suggests that many of the other putative IdeR targets may
behave in a similar fashion (Table 1), but this conclusion
must be validated by further experimentation.
Data presented in this communication and in previous
results (Dussurget et al., 1996; Rodriguez et al., 1999)
show that IdeR is a pleiotropic regulator in M. tuberculosis
and M. smegmatis, making it a functional homologue of
Fur in non-actinomycete bacteria. IdeR has been shown
previously to control siderophore biosynthesis in M.
smegmatis (Dussurget et al., 1996), and we present
evidence that, in M. tuberculosis, IdeR directly affects the
transcription of the mbtA, mbtB and mbtI. It is likely that
other genes in the mbt cluster (mbtA–H, lipK) are co-
transcribed with mbtA, mbtB and mbtI during iron
starvation. Mycobacterial siderophores are synthesized
by non-ribosomal peptide synthases and contain deriva-
tized amino acids (Quadri et al., 1998;De Voss et al., 1999;
2000; Quadri, 2000). As we find IdeR boxes upstream of
genes presumably involved in the biosynthesis of amino
acids such as histidine (hisE, hisB-like) and phenylalanine
( pheA), it is possible that these aromatic amino acids are
also used in siderophores or compounds with siderophore-
like activity. Although IdeRhas been shown to control katG
and sodA indirectly in M. smegmatis, we did not find IdeR
boxes upstream of genes annotated as oxidative stress
detoxifying enzymes; however, a putative IdeR binding
site with six mismatches was identified upstream of sodC
(data not shown).
The mechanism of transcriptional control of iron storage
in bacteria has yet to be completely elucidated. In E. coli, a
putative 5
Fur box was identified upstream of bfrA
(Andrews et al., 1989), and this gene was shown to be
positively regulated by iron (Andrews, 1998). However, no
direct binding of Fur to this putative Fur box has been
observed (Stojiljkovic et al., 1994). bfrA transcription in
Pseudomonas aeruginosa increases during growth in
high-iron medium and also by exposure to hydrogen
peroxide (Ma et al., 1999). ftn, encoding ferritin, another
iron storage molecule in E. coli, is also positively regulated
by Fur (Abdul-Tehrani et al., 1999), but no direct
interaction with Fur has been observed, either. Thus, Fur
can activate the transcription of genes involved in iron
storage, but it is currently unknown whether this effect is
direct or indirect.
Intriguingly, bfrA in M. tuberculosis is controlled by three
promoters, two are IdeR and iron repressed (P
), whereas the other is activated by IdeR and high
levels of iron in the growth medium (P
) (Fig. 5). The
total amount of bfrA mRNA decreases approximately
threefold during iron starvation, but this level is a sum of
the mRNA produced by the P
and P
: during iron
starvation, P
is activated and P
is repressed (Figs 5
and 6B, D and E). Eliminating the IdeR boxes upstream of
bfrA eliminates P
and significantly lowers, but does not
eliminate, P
transcripts, which indicates that P
is a
Fig. 8. Model of IdeRfunction in M. tuberculosis. Our data indicate that
IdeR functions as a repressor of transcription by blocking the 210
region of IdeR-regulated promoters, which blocks RNA polymerase
(RNAP) from binding and initiating transcription. During iron
starvation, IdeR dissociates from its IdeR box sequence, and RNAP
initiates transcription of both bfd and the bfrA P
. In the presence
of iron, IdeR blocks transcription of bfd and bfrA P
and plays an
as yet uncharacterized role in the activation of the bfrA P
860 B. Gold et al.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
true promoter, and its mRNA is extremely unlikely to be a
cleavage product of the P
transcript (Fig. 6C). Although
IdeR and its binding site are necessary for maximal
expression of bfrA, the fact that some bfrA expression is
observed in the absence of IdeRboxes must be explained.
We favour the hypothesis that IdeR, bound to the two IdeR
boxes, contacts and activates RNA polymerase directly to
transcribe bfrA (Fig. 8). It was shown recently that MntR,
another member of the DtxR family, can both positively
and negatively regulate the expression of manganese
uptake genes in Bacillus subtilis (Que and Helmann,
2000). Thus, like Fur, DtxR-like proteins appear to have
positive roles in transcription (Vasil and Ochsner, 1999). In
the absence of the IdeR boxes, IdeR may still contact RNA
polymerase, but less efficiently than when bound to the
IdeR boxes. This hypothesis is currently being tested. Our
data suggest two potential explanations why bfrA mRNA is
produced under both low- and high-iron conditions. The
simplest model is that M. tuberculosis produces low levels
of BfrA during iron starvation to maintain iron homeostasis
during a shift to an iron-replete medium. The second
model is that regulation of iron storage in M. tuberculosis is
post-transcriptional. This is the case in eukaryotes, in
which ferritin mRNA is transcribed in low iron, but
translation is blocked by the binding of an IRP (iron-
regulatory protein, a cis-aconitase) to the 5
IRE of ferritin
mRNA (Klausner et al., 1993). In this model, bfrA mRNA is
expressed during iron starvation to create an mRNA pool,
which can be rapidly translated and allows cells to store
excess iron if a shift to high iron occurs. In support of this
model, reports have shown that aconitases in prokaryotes
can bind mRNA (Tang and Guest, 1999), and M.
tuberculosis has a cis-aconitase (rv1475c ). In addition,
the sodB mRNA half-life was decreased in a fur mutant,
suggesting that Fur has a role in post-transcriptional
regulation (Dubrac and Touati, 2000). We are currently
investigating the possibility of post-transcriptional regu-
lation of bfrA. Preliminary data from our laboratory indicate
that the M. tuberculosis ferritin homologue, bfrB (rv3841),
is also positively regulated by IdeR, and this mechanism is
currently under investigation (data not shown).
Many of the potential IdeR-regulated genes identified in
this study have no obvious relationship to iron metabolism.
The possibility that genes involved in lipid and membrane
biosynthesis (acpP, murB) are regulated by iron raises an
intriguing possibility that M. tuberculosis alters its
membrane structure under iron-limiting conditions, such
as those encountered during infections. This type of
adaptation has been seen in Salmonella, which was
shown to alter its peptidoglycan structure during infection
of epthithelial cells (Quintela et al., 1997). The phago-
somes of Salmonella and Legionella have been shown to
be limiting for iron, estimated at 1 mM (Byrd and Horwitz,
1989; Garcia-del Portillo et al., 1992; Mahan et al., 1995).
There is evidence that M. tuberculosis encounters an iron-
limited environment during intracellular growth, as myco-
bactin is required for growth in macrophages (De Voss
et al., 2000). M. tuberculosis faces low-iron conditions
during infections in mice, as levels of mbtB mRNA
increase <60-fold and bfrA levels decrease 10-fold in
lungs (J. Timm et al., submitted). In addition, M.
tuberculosis virulence was attenuated in mice when the
bacterium contained a mutated dtxR allele that was
previously shown to behave as a constitutive repressor of
C. diphtheriae genes during iron limitation in an E. coli
surrogate system(Sun et al., 1998; Manabe et al., 1999). It
was hypothesized that the mutated DtxR was decreasing
M. tuberculosis iron acquisition during a mouse infection,
although this has not been shown directly (Manabe et al.,
1999). In the work reported here, we describe the
expression of iron- and IdeR-regulated genes during M.
tuberculosis infection of THP-1 macrophages, and these
results suggest that M. tuberculosis faces an iron-limiting
environment in the macrophage. Therefore, many of the
genes identified by the computer search for IdeR boxes
are good candidates for inactivation and subsequent study
of their roles in virulence.
Experimental procedures
Identification of IdeR iron boxes
An initial low-stringency search of the M. tuberculosis genome
using the BLAST program, querying for the DtxR/IdeR binding
sequence, identified one promoter region of the divergently
transcribed genes, hisE–irg2, which was in fact regulated by
IdeR (Rodriguez et al., 1999). In this work, various
combinations of the consensus DtxR iron box were used to
probe the M. tuberculosis H37Rv published sequence (Cole
et al., 1998) with the program TUBERCULIST (http://genolist. (Moszer et al., 1995). The query
sequence used was TWAGGTWAGSCTWACCTWA (where
W¼A or T and S¼G or C), and we allowed for up to five
mismatches and limited the search to within 2250 to 1100bp
of each ORF’s predicted start codon.
Bacterial strains, media and growth conditions
Escherichia coli strains JM109 and XL1-Blue (Stratagene)
were used for cloning and grown in Luria–Bertani (LB) broth.
Wild-type M. smegmatis mc
155 and the M. smegmatis ideR
strain SM3 were grown in Middlebrook 7H9 liquid medium
supplemented with 0.2% glycerol and 0.05% Tween 80, and
M. tuberculosis strain H37Rv was grown in the same medium
with the addition of 0.5%bovine serumalbumin (BSA) fraction
V, 0.2%dextrose and 0.085%NaCl (ADC supplement). When
required, media were supplemented with 20 mg ml
mycin, 10 mgml
kanamycin and 100mgml
ampicillin. To
assay gene expression by mbRT–PCR or primer extension
analysis in low and high iron, M. tuberculosis was grown in
MMT-ADN (minimal medium with 0.05% Tween 80, treated
Identification of IdeR-regulated genes in M. tuberculosis 861
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
overnight with Chelex 100; Bio-Rad) supplemented with either
0 or 50 mM FeCl
. Final concentrations of iron in low-iron
MMT-ADN have been shown previously by atomic absorption
spectroscopy to be in the range of ,1 mM iron (Rodriguez
et al., 1999). In order to achieve conditions of iron starvation,
M. tuberculosis H37Rv was passaged for two generations in
low-iron medium and then diluted to an optical density of 0.05
in the same medium lacking iron or in a high-iron medium.
b-Galactosidase assays
b-Galactosidase assays were performed as described
previously (Miller, 1972; Dussurget et al., 1999). Briefly, M.
smegmatis was grown in LB mediumsupplemented with 0.1%
Tween to mid-late logarithmic phase (OD
which was made iron limiting by the addition of 2,2
to 200mM and grown for an additional 3h.
bfrA–lacZ constructs
A 430bp construct encompassing 2378 to 152 of bfrA was
amplified by PCR, cloned into the vector psM128 and
integrated in single copy into the att site of M. smegmatis wild
type (MC
155) and ideR mutant (SM3) (Dussurget et al.,
1999). A construct was made by deleting the two iron boxes
(2255 to 2199), resulting in bfrADIB–lacZ, which contains an
additional XhoI site. Constructs were verified by sequencing.
IdeR purification
IdeRwas overexpressed in M. smegmatis, and the protein was
purified by techniques similar to those described previously but
with some modifications (Schmitt et al., 1995; Pohl et al., 1999).
The wild-type copy of the M. tuberculosis ideR was cloned
downstream of the L5 mycobacteriophage promoter, P
, and
then placed into pSM232 (pMV261 1 streptomycin/spectino-
mycin resistance cassette), resulting in pSM273. Strain SM84
was created by transforming the ideR mutant, SM3, with
pSM273. Functional IdeR activity was visualized by comple-
mentation of SM3’s derepression of siderophore synthesis on
CAS high-iron plates (Dussurget et al., 1996). A culture of
SM84 was grown to stationary phase in 7H9 medium, and cells
were harvested and resuspended in buffer A (50mM Tris-HCl,
pH7.5, 50mM NaCl, 2mM b-mercaptoethanol) containing a
protease inhibitor cocktail (Complete; Boehringer Mannheim).
Cells were disrupted by passaging three times by French press
at 1200 psi. Cell debris and lipids were removed by 30min
ultracentrifugation at 20000r.p.m. Crude extracts were loaded
onto a nickel –NTA column (Qiagen) and separated by fast
protein liquid chromatography (FPLC) using an imidazole
gradient of 0–100mMin buffer A. IdeR was eluted between 20
and 30mM imidazole. Fractions containing IdeR were pooled
and dialysed against 50mM Tris-HCl, pH7.5, 50mM NaCl,
1mM dithiothreitol (DTT) and 1mM EDTA to remove residual
nickel. A second FPLC purification was performed using anion
exchange on a Source 15Q column (Amersham Pharmacia
Biotech) with 50mM Tris-HCl, pH7.5, 50mM NaCl, 1mM DTT
and an NaCl gradient of 50–500mM. The major IdeR peak
eluted between 65 and 85mM NaCl. IdeR was .99% pure as
verified by Coomassie staining and Western blotting, with a
yield of about 150mg of purified IdeR l
Gel retardation assay
The gel retardation assay was carried out essentially as
described previously (Hamoen et al., 1998; Rodriguez et al.,
1999) with minor modifications. PCRfragments containing the
putative IdeR consensus binding sites were end labelled with
T4 polynucleotide kinase and [g-
P]-ATP. Binding reactions
[20 mM Tris-HCl at pH8, 1 mM DTT, 50 mM KCl, 5mM MgCl
0.05 mg ml
poly-(dI –dC), 0.05 mg ml
BSA and 10%
glycerol] containing <0.1 ng of probe (20000 c.p.m.) were
incubated with purified IdeR (0.06–1 mM) in 20 ml volumes for
30min at room temperature. NiSO
(200 mM) was added to
binding reactions and, when required, to the acrylamide gels
and running buffers. Reactions (15 ml) were loaded without
dye to an 8% polyacrylamide gel containing 40 mM Tris
acetate at pH8. Gels were run at 110V at room temperature,
dried, and bands were visualized by autoradiography.
DNase footprint analysis
Primers corresponding to either the top or bottom strands of
the following promoter regions, mbtA–mbtB, mbtI, rv3402c,
bfrA–bfd, were labelled with T4 polynucleotide kinase and
P]-ATP and then used to PCR amplify the corresponding
fragments. PCR products were ethanol precipitated and then
purified on a 2% agarose gel. Binding reactions with IdeR
were performed as described for the gel retardation assay in
20ml with <100000 c.p.m. probe and 1 mM IdeR. Reaction
volumes were adjusted to 100ml with a Ca
(final concentrations are 2.5 mM CaCl
and 5 mM MgCl
Then, 0.15U of DNase (Promega) was added, and mixtures
were incubated for 1min at room temperature. Reactions
were terminated by the addition of 90 ml of stop solution
(200 mM NaCl, 20mM EDTA, 1% SDS and 100mgml
RNA). Samples were subsequently extracted with phenol –
chloroform, ethanol precipitated and resuspended in a
formamide loading dye. Samples were electrophoresed on a
6% TBE polyacrylamide–urea sequencing gel, dried and
exposed by autoradiography. Maxam–Gilbert A1G sequen-
cing reactions were performed to identify protected regions.
Primer extension analyses
RNA was extracted essentially according to the protocol of
Manganelli et al. (1999). M. tuberculosis H37Rv was grown as
described above in low- and high-iron MM-ADN to late log
phase. Bacterial pellets were resuspended in LETS buffer
(100 mM LiCl, 10mM EDTA, 10 mM Tris, pH7.8, 1% SDS)
and then mixed with an equal volume of phenol –chloroform–
isoamyl alcohol (P:C:IAA; 25:24:1) and broken with a bead
beater. Membranes were removed by centrifugation at
14000 r.p.m. for 15 min, and the resulting supernatant was
extracted once in P:C:IAA and twice with TRI reagent
(Molecular Research Center), according to the manufac-
turer’s protocol. RNA integrity was verified by electrophoretic
analysis on a 2% TBE agarose gel. For primer extension, 20–
60mg of RNA was hybridized to 1.5 pmol of g-
862 B. Gold et al.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
reverse primer in 1Â Carboxydothermus hydrogenoformans
reverse transcription buffer (C. therm. kit; Roche) in a final
volume of 10ml. The hybridization programme was 948C for
1.5 min, 648C for 3min, 578C for 5 min, and then the tubes
were allowed to cool rapidly to 508C, when they were placed
directly on ice. A reverse transcription mixture using the C.
therm reverse transcriptase (Roche; final of 1mM dNTPs,
2.5 mM MgCl
, 5% DMSO and 5mM DTT) was added to the
annealed primer –RNA mixture at a final volume of 20ml. The
cDNA synthesis reaction was at 608C for 1 h followed by 2min
at 948C, and then the cDNA–RNA mixture was ethanol
precipitated and washed with 75% EtOH. Samples were
resuspended in 6 ml of water and 4 ml of formamide loading
buffer and separated on a 6% TBE gel containing 8 M urea at
1800 V. In some cases, a 10 bp ladder (Gibco BRL) was used
as a molecular weight standard to determine the length of the
primer extension product and, in other cases, a sequencing
reaction was performed using the Sequenase 7-deaza-dGTP
kit (USB). All transcriptional start points were verified using
two primers whose 3
ends were 10–20 bp apart.
Molecular beacon RT–PCR (mbRT–PCR)
To obtain RNA, frozen bacterial pellets were resuspended in
1ml of TRI reagent and 500ml of silica beads and then broken
using a bead beater at maximum speed for 2Â1min, with
chilling on ice between breaking periods. RNA extraction was
performed according to the manufacturer’s instructions
(Molecular Research Center). The mbRT–PCR was per-
formed essentially as described by Manganelli et al. (1999;
2001), and cDNA was synthesized as described above for
primer extension assays. A cocktail of up to five reverse
primers was added per reaction to have all reverse
transcriptions (always including sigA) performed in the same
tube. RNA (50–200ng) was added to each reverse transcrip-
tion and, in all cases, a mock reaction lacking reverse
transcriptase was performed to obtain copy numbers of DNA
contaminating the RNA sample. The PCR reactions typically
contained 100–800nM of a gene-specific molecular beacon
(coupled to tetramethylrhodamine or fluorescein) and were
performed with the following PCRprogramme: 958Cfor 10min,
10 cycles of 958C for 20s, 658C for 30s and 728C for 30s, and
then 35 cycles of 958C for 20s, 608C for 30s and 728C for 30s.
Molecular beacons were designed with the aid of the MFOLD
server (,mfold/dna/form1.cgi) and
synthesized as described previously (Tyagi and Kramer, 1996).
All molecular beacon and primer sequences are available upon
request. Ratios of induction at each time point were solved
using the following formula, where X and A are the absolute
number of copies of mRNA from geneX or sigA respectively:
low Fe
low Fe
high Fe
high Fe
Isolation of RNA from THP-1 macrophages infected with
M. tuberculosis
Mycobacterium tuberculosis infections of THP-1-derived
macrophages were performed as described by Manganelli
et al. (2000). Briefly, THP-1 cells were grown in RPMI-1640
(supplemented with 0.45% glucose, 0.15% sodium pyruvate
and 4mM L-glutamine), differentiated into macrophages by
24 h treatment with 50 nM 12-O-tetradecanoylphorbol-
13-acetate (PMA) (Tsuchiya et al., 1982) and plated at a
final concentration of 7.5 Â10
cells ml
. To improve long-
term macrophage adherence in large volumes, flasks were
pretreated with 0.2% gelatin overnight at 48C. M. tuberculosis
H37Rv were grown in 7H9 to an OD
of 0.2 and infected into
the THP-1 cells at a multiplicity of infection of 0.5–1.
Extracellular bacteria were removed after 2h by washing
twice with phosphate-buffered saline (PBS), and then fresh
RPMI containing 50 mgml
gentamicin and 20% fetal calf
serum (FCS) was added back to the macrophages. At 24 and
72 h, RPMI was removed, macrophages were washed once
with RPMI, and then the remaining cells were resuspended in
10 ml of TRI reagent containing polyacryl carrier, frozen
immediately on dry ice and then stored at 2808C. At each time
point, macrophages were checked for viability using Trypan
blue exclusion (Sigma), and viable counts of M. tuberculosis
growing intra- and extracellularly were performed in a parallel
infection. Expression levels of all genes were normalized
against M. tuberculosis that had been exposed to RPMI for 2h
or to an M. tuberculosis rolling culture in 7H9. To make RNA,
500ml of silica beads was added to the frozen mix of M.
tuberculosis and macrophages, and cells were broken using a
bead beater at maximum speed for 2Â1 min, with chilling on
ice between breaking periods. RNA extraction was performed
with TRI reagent according to the manufacturer’s instructions
(Molecular Research Center). RNA was subjected to DNase
treatment (DNA-free kit; Ambion) and then precipitated and
resuspended in water treated with diethylpyrocarbonate
(DEPC). Ratios of induction at each time point were
calculated using the following formula, where X and A are
the absolute number of copies of mRNA from geneX or sigA
respectively: (X
We would like to thank members of the Smith laboratory for
support and fruitful discussions. For help with the M.
tuberculosis gene expression during macrophage infections,
we would like to thank Riccardo Manganelli, Patricia Fontan
and Eugenie Dubnau. For excellent advice regarding protein
purification, gel shifts and DNase footprints, we would like to
thank Leendert Hamoen, Bert Jan Haijema and Kursad
Turgay. This work was supported by grant AI-44865 (awarded
to I.S.) and a UNCF Parke-Davis postdoctoral fellowship
(awarded to G.M.R.) and was in partial fulfilment of the PhD
thesis of B.G., Department of Microbiology, NYU Medical
Note added in proof
The cis-aconitase in M. tuberculosis has an 80% amino
acid identity to the RNA binding motif of the human cis-
aconitase of DLVIDHSIQV. We thank A. L. Sonenshein for
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