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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
macrophages
Benjamin Gold,
1,2
G. Marcela Rodriguez,
2
Salvatore A.
E. Marras,
2
Mickey Pentecost
3
and Issar Smith
1,2
*
1
Department of Microbiology, New York University
Medical Center, New York, NY 10016, USA.
2
TB Center, Public Health Research Institute, 455 First
Avenue, New York, NY 10016, USA.
3
Albert Nerkin School of Engineering, Cooper Union, New
York, NY, USA.
Summary
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.
Introduction
The low levels of available iron in a bacterial pathogen’s
mammalian host (estimated at 10
218
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 smitty@phri.nyu.edu; 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
0
end of the IdeR box to the predicted ORF start site.
852 B. Gold et al.
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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
macrophages.
Results
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
32
P-labelled DNA probes in the presence of 200 mM Ni
21
, and complexes were resolved on an 8% Tris acetate polyacrylamide gel containing
200 mM Ni
21
. 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.
Identification of IdeR-regulated genes in M. tuberculosis 853
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
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.
pasteur.fr/TubercuList/ 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.
32
P-labelled fragments were bound to 1 mM purified IdeR in the presence of 200 mM Ni
21
and
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.
854 B. Gold et al.
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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
databases.
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
boxes
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
32
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
0
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.,
1992).
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
Identification of IdeR-regulated genes in M. tuberculosis 855
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
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
3
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
0
mRNA termini were mapped using
32
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
2
155 harbouring
the M. tuberculosis bfrA–lacZ: (A) P
high
using RNA from cells grown in a medium containing high iron; and (B) P
low1
and P
low2
, using RNA from cells
grown in an iron-limiting medium. The locations of P
low1
, P
low2
and P
high
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
2
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
2
155 and
SM3.
Initial experiments using the intact bfrA–lacZ fusion
integrated into M. smegmatis MC
2
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
low1
, P
low2
and P
high
. The expression of P
high
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
low
expression in high and low iron was analysed only using the
bfrA–lacZ (F), as the deletion of the IdeR boxes abrogates P
low
activity. The regulation of bfrA was analysed by mbRT–PCR using a beacon that
recognizes a sequence common to the mRNAs produced by P
low
and P
high
, and recognizing a sequence 5
0
to P
high
and unique to P
low
(E).
858 B. Gold et al.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
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
P
low1
and P
low2
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
high
) (Figs 5A and 6B). P
high
activity
is decreased during iron starvation (Fig. 6B, lanes 1 and 2)
and is dependent on IdeR, as the levels of P
high
transcript
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
high
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
high
activity (Fig. 6C, lanes 3 and 4). The mRNA produced by
P
high
is not a processed mRNA produced by P
low
, as it is
made, albeit at lower levels, even when P
low
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
low1
and P
low2
) (Figs 5B and 6D).
P
low1
and P
low2
were always present in equal amounts and
will henceforth be named P
low
to refer to both promoters.
The repression of P
low
in high iron is dependent on IdeR,
as the transcription of P
low
remains high in the ideR mutant
(Fig. 6D). The activity of P
high
was estimated to be 5–10
times stronger than P
low
by quantitative RT–PCR and
band intensity from primer extension (data not shown).
The contributions of P
low
and P
high
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
low
1P
high
) decreased in
a time-dependent manner to about 30% during iron
starvation (Fig. 6E). Using RT–PCR with a beacon
specific for the P
low
mRNA (which recognizes a sequence
5
0
to P
high
), P
low
was demonstrated to be induced about
11-fold during iron starvation (Fig. 6E). The decrease in
bfrA mRNA (P
low
1P
high
) during iron starvation can be
attributed to a combination of mRNA differentially
expressed from both P
low
and P
high
(Fig. 8). P
high
is
expressed during growth in conditions of iron excess and
requires IdeR for expression. During conditions of iron
starvation, P
high
is shut off, and IdeR releases its
repression of P
low
, allowing for low-level expression from
P
low
.
IdeR-controlled genes are derepressed during M.
tuberculosis infection of human macrophage-like THP-1
cells
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.
Identification of IdeR-regulated genes in M. tuberculosis 859
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
by 72 h (Fig. 7). The levels of bfrA (P
low
1P
high
) 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).
Discussion
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
0
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
low1
and
P
low2
), whereas the other is activated by IdeR and high
levels of iron in the growth medium (P
high
) (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
low
and P
high
: during iron
starvation, P
low
is activated and P
high
is repressed (Figs 5
and 6B, D and E). Eliminating the IdeR boxes upstream of
bfrA eliminates P
low
and significantly lowers, but does not
eliminate, P
high
transcripts, which indicates that P
high
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
low1,2
. In the presence
of iron, IdeR blocks transcription of bfd and bfrA P
low1,2
and plays an
as yet uncharacterized role in the activation of the bfrA P
high
.
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
low
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
0
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.
pasteur.fr/TubercuList/) (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
2
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
21
strepto-
mycin, 10 mgml
21
kanamycin and 100mgml
21
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
3
. 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
600
¼0.4–0.6),
which was made iron limiting by the addition of 2,2
0
-dipyridyl
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
2
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
left
, 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
21
culture.
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-
32
P]-ATP. Binding reactions
[20 mM Tris-HCl at pH8, 1 mM DTT, 50 mM KCl, 5mM MgCl
2
,
0.05 mg ml
21
poly-(dI –dC), 0.05 mg ml
21
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
4
(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
[g-
32
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
21
/Mg
21
solution
(final concentrations are 2.5 mM CaCl
2
and 5 mM MgCl
2
).
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
21
yeast
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-
32
P-labelled
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
2
, 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
0
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 (http://bioinfo.math.rpi.edu/,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:
(X
low Fe
/A
low Fe
)/(X
high Fe
/A
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
5
cells ml
21
. 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
540
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
21
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
intracellular
/A
intracellular
)/(X
extracellular
/A
extracellular
).
Acknowledgements
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
Center.
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
bringing this to our attention.
References
Abdul-Tehrani, H., Hudson, A.J., Chang, Y.S., Timms, A.R.,
Hawkins, C., Williams, J.M., et al. (1999) Ferritin mutants of
Identification of IdeR-regulated genes in M. tuberculosis 863
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
Escherichia coli are iron deficient and growth impaired, and
fur mutants are iron deficient. J Bacteriol 181: 1415–1428.
Andrews, S.C. (1998) Iron storage in bacteria. Adv Microb
Physiol 40: 281–351.
Andrews, S.C., Harrison, P.M., and Guest, J.R. (1989)
Cloning, sequencing, and mapping of the bacterioferritin
gene (bfr ) of Escherichia coli K-12. J Bacteriol 171:
3940–3947.
Andrews, S.C., Smith, J.M., Hawkins, C., Williams, J.M.,
Harrison, P.M., and Guest, J.R. (1993) Overproduction,
purification and characterization of the bacterioferritin of
Escherichia coli and a C-terminally extended variant. Eur J
Biochem 213: 329–338.
Bearden, S.W., and Perry, R.D. (1999) The Yfe system of
Yersinia pestis transports iron and manganese and is
required for full virulence of plague. Mol Microbiol 32:
403–414.
Byrd, T.F., and Horwitz, M.A. (1989) Interferon gamma-
activated human monocytes down-regulate transferrin
receptors and inhibit the intracellular multiplication of
Legionella pneumophila by limiting the availability of iron.
J Clin Invest 83: 1457–1465.
Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C.,
Harris, D., et al. (1998) Deciphering the biology of
Mycobacterium tuberculosis from the complete genome
sequence (see comments) [published erratum appears in
Nature 1998 November 12; 396 (6707): 190]. Nature 393:
537–544.
De Voss, J.J., Rutter, K., Schroeder, B.G., and Barry, C.E., III
(1999) Iron acquisition and metabolism by mycobacteria. J
Bacteriol 181: 4443–4451.
De Voss, J.J., Rutter, K., Schroeder, B.G., Su, H., Zhu, Y.,
and Barry, C.E., III (2000) The salicylate-derived myco-
bactin siderophores of Mycobacterium tuberculosis are
essential for growth in macrophages. Proc Natl Acad Sci
USA 97: 1252–1257.
Doukhan, L., Predich, M., Nair, G., Dussurget, O., Mandic-
Mulec, I., Cole, S.T., et al. (1995) Genomic organization of
the mycobacterial sigma gene cluster. Gene 165: 67–70.
Dubrac, S., and Touati, D. (2000) Fur positive regulation of
iron superoxide dismutase in Escherichia coli: functional
analysis of the sodB promoter. J Bacteriol 182: 3802–3808.
Dussurget, O., Rodriguez, M., and Smith, I. (1996) An ideR
mutant of Mycobacterium smegmatis has derepressed
siderophore production and an altered oxidative-stress
response. Mol Microbiol 22: 535–544.
Dussurget, O., Rodriguez, M., and Smith, I. (1998) Protective
role of the Mycobacterium smegmatis IdeR against reactive
oxygen species and isoniazid toxicity. Tuber Lung Dis 79:
99–106.
Dussurget, O., Timm, J., Gomez, M., Gold, B., Yu, S., Sabol,
S.Z., et al. I. (1999) Transcriptional control of the iron-
responsive fxbA gene by the mycobacterial regulator IdeR.
J Bacteriol 181: 3402–3408.
Fiss, E.H., Yu, S., and Jacobs, W.R., Jr (1994) Identification of
genes involved in the sequestration of iron in mycobacteria:
the ferric exochelin biosynthetic and uptake pathways. Mol
Microbiol 14: 557–569.
Garcia-del Portillo, F., Foster, J.W., Maguire, M.E., and
Finlay, B.B. (1992) Characterization of the micro-environment
of Salmonella typhimurium-containing vacuoles within
MDCK epithelial cells. Mol Microbiol 6: 3289–3297.
Garg, R.P., Vargo, C.J., Cui, X., and Kurtz, D.M., Jr (1996) A
[2Fe-2S] protein encoded by an open reading frame
upstream of the Escherichia coli bacterioferritin gene.
Biochemistry 35: 6297–6301.
Gomez, M., and Smith, I. (2000) Determinants of Mycobac-
terial gene expression. In Molecular Genetics of Mycobac-
teria. Hatfull, G.F., andJacobs, W.R. (eds). Washington,
DC: American Society for Microbiology Press, pp.
111–129.
Hamoen, L.W., Van Werkhoven, A.F., Bijlsma, J.J., Dubnau,
D., and Venema, G. (1998) The competence transcription
factor of Bacillus subtilis recognizes short A/T-rich
sequences arranged in a unique, flexible pattern along the
DNA helix. Genes Dev 12: 1539–1550.
Hantke, K. (2001) Iron and metal regulation in bacteria. Curr
Opin Microbiol 4: 172–177.
Janakiraman, A., and Slauch, J.M. (2000) The putative iron
transport system SitABCD encoded on SPI1 is required for
full virulence of Salmonella typhimurium. Mol Microbiol 35:
1146–1155.
Klausner, R.D., Rouault, T.A., and Harford, J.B. (1993)
Regulating the fate of mRNA: the control of cellular iron
metabolism. Cell 72: 19–28.
Lee, J.H., Wang, T., Ault, K., Liu, J., Schmitt, M.P., and
Holmes, R.K. (1997) Identification and characterization of
three new promoter/operators from Corynebacterium
diphtheriae that are regulated by the diphtheria toxin
repressor (DtxR) and iron. Infect Immun 65: 4273–4280.
Leffel, M.S., and Spitznagel, J.K. (1975) Fate of human
lactoferrin and myeloperoxidase in phagocytizing human
neutrophils: effects of immunoglobulin G subclasses and
immune complexes coated on latex beads. Infect Immun
12: 813–820.
McKinney, J.D., Honer zu Bentrup, K., Munoz-Elias, E.J.,
Miczak, A., Chen, B., Chan, W.T., et al. (2000) Persistence
of Mycobacterium tuberculosis in macrophages and mice
requires the glyoxylate shunt enzyme isocitrate lyase.
Nature 406: 735–738.
Ma, J.F., Ochsner, U.A., Klotz, M.G., Nanayakkara, V.K.,
Howell, M.L., Johnson, Z., et al. (1999) Bacterioferritin A
modulates catalase A (KatA) activity and resistance to
hydrogen peroxide in Pseudomonas aeruginosa. J Bacter-
iol 181: 3730–3742.
Mahan, M.J., Tobias, J.W., Slauch, J.M., Hanna, P.C., Collier,
R.J., and Mekalanos, J.J. (1995) Antibiotic-based selection
for bacterial genes that are specifically induced during
infection of a host. Proc Natl Acad Sci USA 92: 669–673.
Manabe, Y.C., Saviola, B.J., Sun, L., Murphy, J.R., and
Bishai, W.R. (1999) Attenuation of virulence in Mycobac-
terium tuberculosis expressing a constitutively active iron
repressor. Proc Natl Acad Sci USA 96: 12844–12848.
Manganelli, R., Dubnau, E., Tyagi, S., Kramer, F.R., and
Smith, I. (1999) Differential expression of 10 sigma factor
genes in Mycobacterium tuberculosis. Mol Microbiol 31:
715–724.
Manganelli, R., Tyagi, S., and Smith, I. (2001) Real time PCR
using molecular beacons: a new tool to identify point
mutations and to analyze gene expression in Mycobacter-
ium tuberculosis. In Methods in Molecular Medicine:
864 B. Gold et al.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865
Mycobacterium tuberculosis Protocols, Vol. 54. Parish, T.,
andStoker, N.G. (eds). Totowa, NJ: Humana Press, pp.
295–310.
Miller, C.D., Kim, Y.C., Walsh, M.K., and Anderson, A.J.
(2000) Characterization and expression of the Pseudomo-
nas putida bacterioferritin alpha subunit gene. Gene 247:
199–207.
Miller, J.H. (1972) Experiments in Molecular Genetics. Cold
Spring Harbor, NY: Cold Spring Laboratory Press.
Moszer, I., Glaser, P., and Danchin, A. (1995) SubtiList: a
relational database for the Bacillus subtilis genome.
Microbiology 141: 261–268.
Muller, D., Edwards, M.L., and Smith, D.W. (1983) Changes in
iron and transferrin levels and body temperature in
experimental airborne legionellosis. J Infect Dis 147:
302–307.
Neilands, J.B. (1995) Siderophores: structure and function of
microbial iron transport compounds. J Biol Chem 270:
26723–26726.
Pohl, E., Holmes, R.K., and Hol, W.G.J. (1999) Crystal
structure of the iron-dependent regulator (IdeR) from
Mycobacterium tuberculosis shows both metal binding
sites fully occupied. J Mol Biol 285: 1145–1156.
Pope, C.D., O’Connell, W., and Cianciotto, N.P. (1996)
Legionella pneumophila mutants that are defective for iron
acquisition and assimilation and intracellular infection.
Infect Immun 64: 629–636.
Quadri, L.E. (2000) Assembly of aryl-capped siderophores by
modular peptide synthetases and polyketide synthases.
Mol Microbiol 37: 1–12.
Quadri, L.E., Sello, J., Keating, T.A., Weinreb, P.H., and
Walsh, C.T. (1998) Identification of a Mycobacterium
tuberculosis gene cluster encoding the biosynthetic
enzymes for assembly of the virulence-conferring side-
rophore mycobactin. Chem Biol 5: 631–645.
Quail, M.A., Jordan, P., Grogan, J.M., Butt, J.N., Lutz, M.,
Thomson, A.J., et al. (1996) Spectroscopic and voltam-
metric characterisation of the bacterioferritin- associated
ferredoxin of Escherichia coli. Biochem Biophys Res
Commun 229: 635–642.
Que, Q., and Helmann, J.D. (2000) Manganese homeostasis
in Bacillus subtilis is regulated by MntR, a bifunctional
regulator related to the diphtheria toxin repressor family of
proteins. Mol Microbiol 35: 1454–1468.
Quintela, J.C., de Pedro, M.A., Zollner, P., Allmaier, G., and
Garcia-del Portillo, F. (1997) Peptidoglycan structure of
Salmonella typhimurium growing within cultured mamma-
lian cells. Mol Microbiol 23: 693–704.
Rodriguez, G.M., Gold, B., Gomez, M., Dussurget, O., and
Smith, I. (1999) Identification and characterization of two
divergently transcribed iron regulated genes in Mycobac-
terium tuberculosis [published erratum appears in Tuber
Lung Dis 1999; 79(6): 382]. Tuber Lung Dis 79: 287–298.
Schmitt, M.P., and Holmes, R.K. (1994) Cloning, sequence,
and footprint analysis of two promoter/operators from
Corynebacterium diphtheriae that are regulated by the
diphtheria toxin repressor (DtxR) and iron. J Bacteriol 176:
1141–1149.
Schmitt, M.P., Twiddy, E.M., and Holmes, R.K. (1992)
Purification and characterization of the diphtheria toxin
repressor. Proc Natl Acad Sci USA 89: 7576–7580.
Schmitt, M.P., Predich, M., Doukhan, L., Smith, I., and
Holmes, R.K. (1995) Characterization of an iron-dependent
regulatory protein (IdeR) of Mycobacterium tuberculosis as
a functional homolog of the diphtheria toxin repressor
(DtxR) fromCorynebacteriumdiphtheriae. Infect Immun 63:
4284–4289.
Stojiljkovic, I., Baumler, A.J., and Hantke, K. (1994) Fur
regulon in gram-negative bacteria. Identification and
characterization of new iron-regulated Escherichia coli
genes by a fur titration assay [published erratum appears in
J Mol Biol 1994 Jul 15; 240(3): 271]. J Mol Biol 236:
531–545.
Sun, L., vanderSpek, J., and Murphy, J.R. (1998) Isolation
and characterization of iron-independent positive dominant
mutants of the diphtheria toxin repressor DtxR. Proc Natl
Acad Sci USA 95: 14985–14990.
Tang, Y., and Guest, J.R. (1999) Direct evidence for mRNA
binding and post-transcriptional regulation by Escherichia
coli aconitases. Microbiology 145: 3069–3079.
Tsuchiya, S., Kobayashi, Y., Goto, Y., Okumura, H., Nakae,
S., Konno, T., and Tada, K. (1982) Induction of maturation
in cultured human monocytic leukemia cells by a phorbol
diester. Cancer Res 42: 1530–1536.
Tyagi, S., and Kramer, F.R. (1996) Molecular beacons:
probes that fluoresce upon hybridization. Nature Biotechnol
14: 303–308.
Vasil, M.L., and Ochsner, U.A. (1999) The response of
Pseudomonas aeruginosa to iron: genetics, biochemistry
and virulence. Mol Microbiol 34: 399–413.
Weinberg, E.D. (1984) Iron withholding: a defense against
infection and neoplasia. Physiol Rev 64: 65–102.
White, A., Ding, X., vanderSpek, J.C., Murphy, J.R., and
Ringe, D. (1998) Structure of the metal-ion-activated
diphtheria toxin repressor/tox operator complex. Nature
394: 502–506.
Wright, D.G., and Gallin, J.I. (1979) Secretory responses of
human neutrophils: exocytosis of specific (secondary)
granules by human neutrophils during adherence in vitro
and during exudation in vivo. J Immunol 123: 285–294.
Yu, S., Fiss, E., and Jacobs, W.R., Jr (1998) Analysis of the
exochelin locus in Mycobacterium smegmatis: biosynthesis
genes have homology with genes of the peptide synthetase
family. J Bacteriol 180: 4676–4685.
Yuan, Y., Crane, D.D., Simpson, R.M., Zhu, Y.Q., Hickey,
M.J., Sherman, D.R., and Barry, C.E., III (1998) The 16-kDa
alpha-crystallin (Acr) protein of Mycobacteriumtuberculosis
is required for growth in macrophages. Proc Natl Acad Sci
USA 95: 9578–9583.
Identification of IdeR-regulated genes in M. tuberculosis 865
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851–865