Here we demonstrate through electromobility shift assay EMSA and DNase I footprinting that CmtR cooperatively interacts with multiple binding sites and protects a 90 bp sequence in the C
Trang 1cooperatively interacts with multiple operator
sites to autorepress its transcription in
Mycobacterium tuberculosis
Santosh Chauhan, Anil Kumar, Amit Singhal, Jaya Sivaswami Tyagi and H Krishna Prasad
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
All organisms require metal ions as cofactors for
several enzymatic reactions The physiological
concen-trations of metal ions are maintained by the
coordi-nated action of a family of intracellular metal-sensing
and transporter proteins [1,2] One such family of
metal-sensor proteins (SmtB⁄ ArsR) functions
exclu-sively as transcriptional repressors by regulating
intra-cellular metal ion concentrations under conditions of
surplus metal ions [2] These repressors sense di- and
multivalent heavy metal ions and regulate the
expres-sion of gene(s) encoding protein(s) that specifically expel or chelate metal ion(s) [2] These metalloregula-tory repressors bind the operator⁄ promoter DNA regions in operons regulated by stress-inducing concen-trations of heavy metal ions Protein binding to the operator⁄ promoter DNA is strongly inhibited by metal binding to the sensing apoprotein A number of these metal-responsive transcriptional regulatory proteins have been described in a variety of microbes, namely SmtB, a Zn2+-responsive transcriptional repressor in
Keywords
ArsR–SmtB repressor; autoregulation;
CmtR; metalloregulatory repressors;
Rv1994c
Correspondence
H K Prasad, TB Immunology Laboratory,
Department of Biotechnology, All India
Institute of Medical Sciences, New Delhi
110029, India
Fax: +91 11 26589286
Tel: +91 11 26594994
E-mail: hk_prasad@hotmail.com
J S Tyagi, TB Molecular Biology
Laboratory, Department of Biotechnology,
All India Institute of Medical Sciences,
New Delhi 110029, India
Fax: +91 11 26588663
Tel: +91 11 26588491
E-mail: jstyagi@aiims.ac.in
(Received 27 January 2009, revised 4 April
2009, accepted 20 April 2009)
doi:10.1111/j.1742-4658.2009.07066.x
CmtR is a repressor of the ArsR–SmtB family from Mycobacterium tuber-culosis that has been shown to sense Cd(II) and Pb(II) in Mycobacte-rium smegmatis We establish here that CmtR binds cooperatively to multiple sites in M tuberculosis DNA and protects an unusually long
90 bp AT-rich sequence from )80 bp to +10 with respect to its own initiation codon CmtR interacts with four hyphenated imperfect inverted repeats matching the consensus sequence TA⁄ GTAA-N4–5-TT⁄ GATA in the protected region SDS–PAGE and formaldehyde crosslinking experi-ments showed that CmtR forms higher-order oligomers (up to an octamer) The oligomerization of CmtR is in agreement with the cooperative binding
of CmtR to multiple sites on DNA Two promoters transcribe cmtR, and the major promoter physically overlaps with CmtR binding sites Autorepression of CmtR is mediated by cooperative interaction of CmtR with multiple sites on DNA that occlude the major operon promoter The combined results of a GFP reporter assay, an electromobility shift assay and a DNase I footprinting experiment establish that Cd(II), not Pb(II), disrupts the interaction of CmtR with DNA to de-repress transcription of the cmtR–Rv1993c–cmtA operon in M tuberculosis
Abbreviations
EMSA, electromobility shift assay; TSP, transcription start point.
Trang 2Synechococcusspecies [3], ArsR in Escherichia coli [4],
ZiaR in Synechocystis species [5], MerR in
Streptomy-ces lividans[6] and CadC in Staphylococcus aureus [7]
Mycobacterium tuberculosis, a versatile pathogen,
sur-vives in a variety of harsh environmental conditions
including the phagosome of the mammalian host cell
Inside the phagosome, M tuberculosis must adapt to
metal fluxes and modulate the expression of genes
involved in metal detoxification and efflux Ten putative
SmtB⁄ ArsR metal sensors have been identified in the
M tuberculosis genome [8], of which three – namely
NmtR [9], CmtR [10–12] and KmtR [13] – have been
partially characterized CmtR from M tuberculosis is a
winged helical DNA-binding repressor that was shown
to sense cadmium and lead in the surrogate host
Myco-bacterium smegmatis and is proposed to bind a single
25 bp site in the cmtR operator⁄ promoter region in the
form [10] The cadmium–CmtR complex and
apo-CmtR were both shown to exist as a homodimer [12]
CmtR was also shown to be upregulated (approximately
threefold) upon entry into macrophages [14]
Cadmium is present as an air pollutant and in
ciga-rette smoke [15] Cadmium is known to accumulate in
human aleovolar macrophages [15] M tuberculosis
may be exposed to toxic concentrations of cadmium in
macrophages As CmtR senses cadmium, it may
mod-ulate the expression of genes involved in detoxification
and efflux of this toxic metal from the mycobacterium
Here we demonstrate through electromobility shift
assay (EMSA) and DNase I footprinting that CmtR
cooperatively interacts with multiple binding sites and
protects a 90 bp sequence in the CmtR operator⁄ pro-moter region Consistent with these data, we show here that CmtR exists as multimers under non-reducing conditions The combined results from EMSA, DNase
I footprinting, transcription start point (TSP) mapping and a GFP reporter assay showed that CmtR represses its transcription by promoter occlusion, and that Cd(II) dislodges CmtR from the operator⁄ promoter to de-repress transcription of the cmtR–Rv1993c–cmtA operon
Results
Co-transcription of cmtR, Rv1993c and cmtA in
M tuberculosis cmtR is located in the proximity of Rv1993c and Rv1992c⁄ cmtA ⁄ ctpG in the M tuberculosis genome (Fig 1A) cmtR was previously shown to be co-tran-scribed with Rv1993c and cmtA in M bovis [10] To establish that cmtR, Rv1993c and cmtA are co-tran-scribed and constitute an operon in M tuberculosis, RT-PCR was performed with logarithmic-phase RNA using primers 94RTf and 94RTr (Fig 1A) The antici-pated PCR product of 330 bp was detected (Fig 1B, lane 2)
Purification of recombinant CmtR protein
M tuberculosis CmtR protein with an N-terminal hexahistidine tag (His-CmtR) was overexpressed in
156 bp
Rv1993c Rv1992c/cmtA/ctpG
94RTf 94RTr
1 2 3
330 bp
16 12.5
1 2
anti-CmtR
Dimer Dimer
1 2
anti-His
16
Dimer Trimer
A
Fig 1 (A) Schematic representation of the cmtR–1993c–cmtA gene locus in M tuberculosis (B) Co-transcription of cmtR–1993c–cmtA genes in M tuberculosis RT-PCR products of RNA from cultures of M tuberculosis H37Rv were amplified using primers 94RTf and 94RTr Lane 1, negative control (without reverse transcriptase); lane 2, cDNA; lane 3, genomic DNA (amplification control) (C) Immunoblot analysis
of purified CmtR Nitrocellulose blots were probed using anti-CmtR (C) and anti-His (D) serum Lane 1, His-CmtR; lane 2, CmtR Dimer and trimer species are indicated by arrowheads The molecular mass of bands (in kDa) as predicted based on the use of a protein molecular mass marker is indicated.
Trang 3E coli The fusion protein was purified by Ni-chelate
affinity chromatography, and was determined to be
approximately 16 kDa in western blots developed
with anti-CmtR serum or anti-His monoclonal IgG
(Fig 1C,D, lane 1) The histidine tag of His-CmtR
was removed using recombinant Tobacco etch virus
(rTEV) protease to give a protein of
approxi-mately 12.5 kDa, CmtR (Fig 1C, lane 2)
Further-more, proteins corresponding to dimers (32 and
25 kDa) and a trimer (48 kDa) were also detectable
(Fig 1C,D)
Mapping of in vivo transcription initiation sites
Two TSPs were identified upstream of cmtR by
primer extension analysis (Fig 2A) The major primer
extension product (T1cmtR) was identified, starting
with the ‘G’ nucleotide located 34 bp upstream of the
CmtR translational start site (Fig 2A,B) Another
primer extension product (T2cmtR), which was
rela-tively weak, was also identified, starting with the ‘G’
nucleotide located 111 bp upstream of CmtR
transla-tional start site (Fig 2A,B) The putative )10
promoter elements identified upstream of T1cmtR
(con-served at five of the six positions) and T2cmtR
(conserved at three of the six positions) showed a
resemblance to the SigA )10 element (Fig 2B), but
)35 elements of both the promoters were modestly
conserved (two of the six positions, SigA consensus
sequence TTGACW-N17-TATAMT where W = A⁄ T,
M = A⁄ C [16])
CmtR interacts with a 90 bp sequence spanning the translational start site
To map the CmtR binding site precisely, DNase I footprinting was performed using purified CmtR and
315 bp of DNA that includes the sequence from )191 bp to +124 bp with respect to the translational start site of CmtR An unusually long 90 bp protected region was observed, which spans )80 bp to +10 bp with respect to translational start site of CmtR (Figs 3 and 4A) This result suggests that CmtR binds to mul-tiple sites and may interact as a functional multimer in the operator⁄ promoter region Two strong hypersensi-tive sites were observed at intervals of 18 bp from the 3¢ end of the footprint (Fig 3), suggesting that this region of DNA is bent or distorted upon CmtR binding The T1cmtR TSP exactly overlaps the CmtR binding site, and T2cmtR is present upstream of the CmtR binding site (Fig 4A), which indicates that CmtR represses the cmtR–Rv1993c–cmtA operon by obstructing contact between the RNA polymerase and the promoter sequence
CmtR cooperatively interacts with multiple sites
in the cmtR promoter region
A close examination of the CmtR protected sequence revealed four hyphenated inverted repeats matching con-sensus sequence TA⁄ GTAA-N4-5-TT⁄ GATA (Fig 4A,B), which could be the binding site of CmtR EMSA assays were performed with various size fragments of
CmtR Rv1995 –10
T2cmtR (+1)
–10 –35
–35
A
T1cmtR T2cmtR
T1cmtR (+1)
A
B
Fig 2 TSP mapping (A) Primer extension
of M tuberculosis RNA isolated from an aerobic culture (lane 1) Two TSPs were mapped The experiment was repeated with the same results using two different sam-ples of RNA The results of dideoxy sequencing reactions using the same primer are shown on the left (B) Sequence encom-passing both TSPs and the putative )10 and )35 promoter sequences.
Trang 4the operator⁄ promoter region to assess CmtR binding
to these sites (Fig 5A) Four differently migrating
DNA–protein complexes were observed (C1, C2, C3
and C4) when the EMSA was performed with the
larg-est F-1 fragment ()191 to +124 bp with respect to
translational start site of CmtR, Fig 5B), which may
represent binding of CmtR to four different sites
(labeled as sites 1, 2, 3 and 4, Fig 4A) Interestingly,
at least three of these complexes appeared at the
low-est CmtR concentration (Fig 5B), indicating that
CmtR has high affinity for these sites This was
consis-tent with the nearly complete protection observed even
at the lowest protein concentration in DNase I
foot-printing Cooperativity in binding can be assessed from
the breadth of transition on a log plot For
non-coop-erative interactions, an increase of 1.81 log units in
protein concentration is required to increase the bound
fraction from 10% to 90% [17] Positive cooperativity
reduces this span; an increase of 0.5 log units of CmtR
protein concentration (480 nm to 2.4 lm, Fig 5B)
increased the total bound fraction from 10% to 90%
(Fig 5C), which shows a cooperative interaction of CmtR with the cmtR operator⁄ promoter region The DNA–protein complexes were specific as they dissoci-ated in the presence of an excess of cold probe and were conserved in the presence of an excess of non-specific DNA [poly(dI-dC), Fig 5F]
To map CmtR binding sites within the 90 bp pro-tected region, EMSA was performed with a smaller fragment F-2 ()67 to +124 bp relative to the CmtR translational start site) that lacks 13 bp from the 5¢ end of the 90 bp CmtR protected sequence and also the first half of site 1 (Figs 4A and 5A) Only three DNA–protein complexes (C2, C3 and C4) were observed (Fig 5D), suggesting that deletion had indeed disrupted one of the binding sites EMSA with the F-3 fragment ()33 to +124 bp), which include sites 3 and
4, showed only two retarded complexes (C3 and C4) (Fig 5E) Previously, it was shown that CmtR binds
to a single site in the region that spans )33 to )9 bp [10], which includes site 3 and part of site 4 (Fig 4A) Taken together, the results show that CmtR interacts with four binding sites in the 90 bp protected region, which corresponds fairly well to four predicted binding sites in this region To establish that the predicted sites are genuine CmtR binding sites, we performed EMSA with oligonucleotides of approximately 20 bp carrying the predicted binding sites 1–4 (Fig 5G) The EMSA results clearly show that CmtR protein interacts with each of these binding sites, but with low affinity No interaction was observed with non-specific oligonucleo-tide (Fig 5G) The decreased affinity of CmtR to DNA (carrying individual sites) could be attributed to
a loss of cooperativity
CmtR forms dimer and higher-order oligomers Several repressors, e.g EthR [18] and RstR [19] that bind at tandem sites, interact as multimers CmtR interacts with multiple binding sites in the opera-tor⁄ promoter region Two experiments were performed
to examine the oligomeric status of CmtR First, SDS–PAGE analysis of soluble His-CmtR under non-reducing condition shows that it forms dimers (approx-imately 32 kDa) and higher-order oligomers (Fig 6A,B) Bands corresponding to positions up to
an octamer (approximately 48, 64, 80, 96, 112 and
128 kDa) were detected in western blots probed with anti-His (lane 2, Fig 6A) and anti-CmtR serum (data not shown) Similar results were obtained when CmtR from which the His tag had been removed was used (not shown) Under reducing conditions, only mono-mers and dimono-mers of CmtR were observed Interestingly, CmtR is partially dimeric even in the presence of
*
T
A G C
CmtR
0 4.8 7.2 9.6 12
T2cmtR
T1cmtR
ATG
(µ M )
Fig 3 CmtR interacts with a 90 bp sequence in the cmtR–Rv1995
intergenic region DNase I footprinting was performed using cmtR–
Rv1995 intergenic DNA (a 315 bp fragment amplified using primers
P6 and 1994intR; the cmtR non-coding strand was labeled) and
increasing concentrations of CmtR protein Arrowheads (right side)
indicate the hypersensitive sites and the protected region is
indi-cated by a black box The results of dideoxy sequencing reactions
using the same primer and DNA template are also shown Asterisk
indicates the strand which is labelled.
Trang 520 mm dithiothreitol (Fig 6B) or 7.5%
b-mercapto-ethanol (Fig 6A) Second, a formaldehyde crosslinking
experiment was performed to confirm the
oligomeriza-tion of CmtR Although not all oligomeric states were
observed, bands corresponding to the position of a
dimer (approximately 32 kDa) and an octamer
(approximately 128 kDa, Fig 6C) were apparent,
showing that CmtR has an inherent ability to form
oligomers
CmtR is a Cd sensing repressor in M tuberculosis
It has previously been shown that M tuberculosis
CmtR is a cadmium- and lead-sensing repressor in the
surrogate host M smegmatis [10] In order to establish
which metal ion(s) the cmtR promoter is responsive to,
a 212 bp DNA fragment (P6–P3 region, Fig 4) with
156 bp of the cmtR–Rv1995 intergenic region was fused to a promoterless GFP gene in plasmid pFPV27
to give pCmtR, and electroporated into M tuberculosis H37Rv The culture was grown to mid-logarithmic phase (attenuance at 595 nm of approximately 0.3), and subsequently diluted to an attenuance at 595 nm
of 0.1 Metal ions CdCl2, NiCl2, CoCl2 and Pb(NO3)2
were added to the culture at maximum permissive con-centrations as used previously [10] No inhibition of growth was observed in the presence of any of the metal ions used (Fig 7A, inset) Increased GFP flores-cence (approximately 2.5-fold) was observed on addi-tion of Cd(II) but not with any other metal ion tested (Fig 7A) This shows that CmtR senses only Cd(II) but not Pb(II) in M tuberculosis
CmtR
(+1)
Rv1995 P6
P4
1994 intR P3
(–43)
–10 –35
T1cmtR
P8
–35
(–43) 1
2 3 4 (+47)
(+47)
(+1)
A
B
C
Fig 4 (A) Nucleotide sequence and salient features of the cmtR–Rv1995 intergenic region The TSPs (T1cmtRand T2cmtR) are indicated by angled arrows The putative )10 and )35 promoter elements are indi-cated by dashed boxes The CmtR DNase I-protected sequence ( )43 to +47 bp) is boxed Full arrows indicate the CmtR recog-nition sites 1, 2, 3 and 4 The positions of primers are indicated by half-headed arrows The arrowheads indicate hypersensitive sites (B) The sequences of four putative CmtR binding sites and the consensus sequence with which CmtR may interact (C) Detailed map of the intergenic region The four CmtR binding sites (1, 2, 3 and 4) are indicated by white boxes within the
90 bp CmtR recognition sequence (gray box) The TSPs mapped in this study are shown by angled arrows The putative )10 and )35 promoter elements are indicated by small black boxes Primers P3 and P6 were used to amplify the cmtR promoter DNA cloned in GFP reporter vector DNase I foot-printing was performed using P6 and the 1994intR amplicon Primers P6, P4, P8 and 1994intR were used for EMSA.
Trang 6Cadmium disrupts the CmtR–DNA
sequence-specific interaction
The effect of metal ions on in vitro binding of CmtR
to the operator⁄ promoter region was determined using
DNase I footprinting and a gel shift assay Ni(II), Co(II) and Pb(II) were not able to dissociate the com-plex even at high concentrations of 500, 200 and
50 lm, respectively (Fig 7B), whereas Cd(II) disrupted the interaction of CmtR with DNA (Fig 7B,C)
[CmtR] µ M
0.01 0.1 1 10 0
10 20 30 40 50 60 70 80 90 100
C1
C2
F
CmtR
C4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
F-1
CmtR
1 2 3 4 5 6 7 8 9 10
C2
F
C4
F-2
C3 C4
F
CmtR
1 2 3 4 5 6 7 8 9 10 11
F-3
F-1 (315 bp, –191 to +124) F-2 (191 bp, –67 to +124)
F-3 (157 bp, –33 to +124)
CmtR
1 2 3 4
2 3 4
3 4 P8
1994intR
P4
P6
T1
1 2 3 4 5
Site 2 Site 1 Site 3 Site 4
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Control
A
D
G F
E
Fig 5 CmtR binds cooperatively to multiple sites in the cmtR promoter region (A) Various fragments used in EMSA Angled arrows indi-cate the CmtR TSP and the ATG site Primers used to amplify fragments are indiindi-cated by half-headed arrows The putative CmtR binding sites (1, 2, 3, and 4) are indicated (B) 32 P-labeled cmtR promoter DNA F-1 was incubated in the absence (lane 1) or presence (lanes 2–18)
of increasing concentrations of CmtR protein The arrowhead indicates the position of free DNA, and the arrows indicate the DNA–protein complexes The arrow within the gel indicates the position of DNA–protein complex 2 (C) The fraction of bound F-1 DNA (estimated by subtracting free DNA from input DNA, Fig 1A) versus CmtR concentration was plotted using SIGMAPLOT 2001 (www.sigmaplot.com) (D, E)
32 P-labeled F-2 (D) and F-3 (E) fragments were incubated in the absence (lane 1) or presence (lanes 2–10 ⁄ 11) of increasing concentrations of CmtR protein (l M ) (F) A competition assay was performed with the F-1 fragment and 3 l M of CmtR with no competitor (lane 2), with 50 · non-specific competitor [poly(dI-dC), lane 3], or with 10· (lane 4) and 50· (lane 5) self-competitor; lane 1 contains labeled DNA only (G) EMSA was performed with approximately 20 bp double-stranded DNA carrying or not carrying (control) a CmtR binding site (1, 2, 3 and 4) in the absence (lane 1) and presence of 1 l M (lane 2) or 2 l M (lane 3) of CmtR protein Control, 38 bp double-stranded DNA known to bind to the DevR protein of M tuberculosis (unpublished result).
Trang 7DNase I footprinting in the presence of metal ions
confirmed that the site-specific interaction of CmtR
was only dislodged by Cd(II) Together, the in vitro
interaction studies and in vivo reporter assay shows
that Cd(II) dissociates bound CmtR from DNA to
de-repress transcription from the cmtR promoter
Discussion
Intracellular concentrations of metals can be toxic to bacteria; therefore their uptake is tightly regulated by metal-dependent transcription regulators CmtR is a Cd(II)-sensing repressor that may regulate genes involved in reducing the intracellular level of Cd(II)
We demonstrate here by DNase I footprinting and EMSA that CmtR interacts cooperatively with multiple binding sites that span 90 bp of sequence up- and downstream of its own translational start site ()80 to +10 bp) Our results show that CmtR interacts with four imperfect hyphenated inverted repeats matching the consensus sequence TA⁄ GTAA-N4-5-TT⁄ GATA Previously, on the basis of EMSA results, Cavet et al (2003) proposed that CmtR may interact with a single degenerate 10-5-10 hyphenated inverted repeat [10]; however, no such instance of this predicted site was observed within the 90 bp CmtR protected sequence as shown in Fig 4 However, this binding site does not resemble the sites described in the current study In the EMSA assays performed by Cavet et al., the DNA–protein complexes were visualized by ethidium bromide staining [10], which is less sensitive compared
to the radiolabeling and imaging techniques used in the present study Hence they may not have visualized the multiple DNA–protein complexes observed in the pres-ent study In addition, DNase I footprinting experi-ments show that CmtR binds to an extended 90 bp DNA sequence compared to the 25 bp sequence proposed by Cavet et al (2003) To our knowledge, this is the first report of an ArsR–SmtB family repres-sor producing an exceptionally long footprint on DNA and interacting with multiple sites Most of the SmtB⁄ ArsR repressors have been proposed to recog-nize one or two degenerate AT-rich inverted repeats in their operator⁄ promoter region [20–22] ZntR, a SmtB⁄ ArsR family repressor, has been proposed to interact with a hyphenated 9-2-9 inverted repeat (ATA TGAACA-AA-TATTCATAT) within the 49 bp of protected DNA [20] SmtB has been proposed to inter-act with two hyphenated imperfect inverted repeats (6-2-6, TGAACA-GT-TATTCA and 7-2-7, CTGAA TC-AA-GATTCAG) in the smtB operator⁄ promoter region [22]
Primer extension analysis identified two TSPs for cmtR The major TSP (T1cmtR) and the putative )10 and )35 promoter elements completely overlap the CmtR binding sites, and the other TSP (T2cmtR) is present upstream of the CmtR binding sites This architecture of the promoter suggests that the interac-tion of the RNA polymerase is hindered by the
14
29
100
M
50
60
70
200
Monomer
Dimer Trimer Tetramer Pentamer Hexamer
Heptamer Octamer
14
29
97
Monomer Dimer
Higher oligomer (~octamer)
M
Monomer
Dimer
Higher
oligomer
Trimer
Tetramer
A
B
C
Fig 6 CmtR forms oligomers Western blot analysis of His-CmtR
probed with anti-His serum CmtR was boiled in sample buffer in
(A) the absence (lane 2) or presence of 7.5% b-mercaptoethanol
(lane 1), or (B) the absence (lane 1) or presence of increasing
con-centrations of dithiothreitol (1, 5, 10 and 20 m M , lanes 2–5,
respec-tively) and resolved on 12% SDS–PAGE (C) A crosslinking
experiment with CmtR was performed in the absence (lane 1) or
presence (lanes 2 and 3) of 0.1% formaldehyde Lanes 1 and 3
contain 30 lg of CmtR, and lane 2 contains 20 lg of CmtR ‘M’
represents molecular mass markers in kDa.
Trang 8binding of CmtR, resulting in repression of the
cmtR–Rv1993c–cmtAoperon
The CmtR binds cooperatively to multiple sites in a
stepwise manner (in the EMSA) CmtR oligomers up
to an octamer were observed Hence, it may be
possi-ble that CmtR oligomerizes in a stepwise manner on
DNA as has been reported for other repressors [18,19]
All repressors of the SmtB⁄ ArsR family, e.g CadC,
SmtB and ArsR [23–25], have been shown to exist as
homodimers The formation of higher oligomers has
not been observed previously among SmtB⁄ ArsR
fam-ily repressors, but is not uncommon for repressors
gen-erally This property may allow the repressors to
efficiently mask the promoter from RNA polymerase
CmtR was previously shown to exist as a dimer, but
as dithiothreitol was used (1-5 mm) during protein
purification and anaerobic conditions were used during
further experimentation [11,12], it is possible that
reducing conditions may have disrupted the
higher-order oligomers to produce the dimers observed here (Fig 6B) CmtR has six cysteine residues, Cys4, Cys24, Cys35, Cys57, Cys61 and Cys102, of which Cys57, Cys61 and Cys102 bind cadmium ions [11,12] and were shown to be important for cadmium-depen-dent activation [10] Mutation of Cys24 of CmtR made the repressor non-functional, indicating that this resi-due may be involved in oligomerization of CmtR
Our in vivo GFP reporter assay suggests that CmtR binds to Cd(II) and induces cmtR expression in
M tuberculosis by derepression This is in partial agreement with a previous report that Cd(II) and Pb(II) can both act as an inducer of cmtR in
M smegmatis [10] Our in vitro experiments (EMSA and DNase I footprinting) also support the in vivo results, where the sequence-specific interaction was abolished by Cd(II) at physiological concentration (5 lm), but not by Pb(II) even at higher concentra-tions (50 lm) The dissociation of the DNA–protein
100
200
300
400
500
600
700
Time (h)
Control
A
B C
0.5 0.9
0 48 96 144 Time (h)
e 595
Metal ion – – Ni Cd Co Pb
CmtR – +
Metal ion – –
Cd Pb
+ + + + + + + +
F
Fig 7 (A) CmtR is a Cd-sensing repressor
in M tuberculosis CmtR-directed GFP
fluo-rescence in aerobic shaken M tuberculosis
cultures in the absence (control) or presence
of metal ions (Ni, Cd, Co and Pb) at
maximum permissive concentrations GFP
fluorescence is expressed as relative
fluorescence unit (RFU) ⁄ attenuance after
background subtraction The mean values
for two independent experiments are
plot-ted Growth curves for all the strains are
shown in the inset The metal ions were
added to the culture when the attenuance
at 595 nm was 0.1, and GFP fluorescence
was measured at 24 h intervals (B) DNase I
footprinting was performed using fragment
F-1 and 2.4 l M CmtR protein in the absence
and presence of metal ions as indicated.
Ni(II), Cd(II), Co(II) and Pb(II) were added at
concentrations of 500, 5, 200 and 50 l M ,
respectively (C) EMSA with fragment F-2
and CmtR protein (3 l M ) in the absence
(lane 2) or presence of increasing
concentra-tions of Cd(II) (1, 2.5, 5 and 10 l M , lanes
3–6, respectively) and Pb(II) (5, 10, 20 and
40 l M , lanes 7–10, respectively).
Trang 9complex at physiological concentrations shows that
Cd(II) has very high affinity for CmtR The disruption
of a repressor–promoter complex at physiological
con-centration is rare, but was also observed in case of
ZntR [20] The difference between our results and the
previous results [10] could be because of the different
hosts used (M tuberculosis versus M smegmatis),
which may differ in the regulation of genes [26–28]
M smegmatis, which is a non-pathogenic saprophytic
bacteria, may encounter varied metal toxicity
com-pared to pathogenic M tuberculosis, which may
result in a different response to ions Moreover, the
homolog of M tuberculosis CmtR in M smegmatis
(MSMEG_5603) has only 67% similarity with
M tuberculosisCmtR (data not shown)
The cmtR, Rv1993c and cmtA genes constitute an
operon The role of Rv1993c and cmtA is not known,
but the cmtA gene product has sequence similarity to
the well-characterized metal transporting P1-ATPase
pump, which pumps out metal ions that may otherwise
be toxic to the bacterium Our results show that CmtR
binds to multiple sites to repress the operon when the
Cd(II) ion is not present (Fig 8) These sites, which
overlap the major promoter (T1) and are located
downstream of the T2 promoter, do not allow
interac-tion of RNA polymerase with promoter DNA in the
presence of bound CmtR When present, the Cd(II)
ion binds to CmtR and decreases its affinity for DNA,
resulting in its release from DNA and hence
transcrip-tion of the operon (Fig 8) The increased
concentra-tion of CmtA may actively pump the Cd(II) out In
the absence of Cd(II), the cmtR–Rv1993c–cmtA operon
is repressed again by CmtR (Fig 8)
Experimental procedures
Plasmids, bacterial strains and culture conditions
M tuberculosisH37Rv was cultured in Dubos medium con-taining 0.05% Tween-80 plus 0.5% albumin⁄ 0.75% dex-trose⁄ 0.085% NaCl at 37 C under aerobic shaking conditions (220 r.p.m.) E coli DH5a was grown in Luria– Bertani medium usually and in 2· YT medium [29] for pro-tein overexpression Antibiotics, when required, were used
at the concentrations indicated: ampicillin at 100 lgÆmL)1 and kanamycin at 25 lgÆmL)1 All cloning steps were per-formed as described [29] The plasmids and primers used in this study are listed in Tables 1 and 2, respectively
Cloning and purification of CmtR The cmtR coding sequence was amplified from M tubercu-losisH37Rv DNA using primers P1 and P2 (Table 2) engi-neered to contain restriction sites for BamHI and HindIII, respectively The amplified product was digested with the indicated restriction enzymes, and cloned into pPROEx-HTb (Invitrogen, Carlsbad, CA, USA) generating pPRO-CmtR The construct was verified by DNA sequencing Recombinant His-CmtR (an N-terminally histidine-tagged fusion protein of approximately 16.0 kDa) was over-expressed by growing recombinant E coli DH5a at 37C
to an attenuance at 595 nm of 0.4–0.5, followed by induc-tion with 1 mm isopropyl thio-b-d-galactoside for 4 h at
37C The induced cells were harvested, resuspended in buffer A (20 mm Tris pH 8.0, 500 mm NaCl, 20 mm imid-azole, 10% glycerol and 1 mm phenylmethanesulfonyl fluo-ride) and sonicated (Branson Ultrasonics, Danbury, CT, USA) on ice (duty cycle 60, four pulses of 2 min each) The
–10 –35
T1 T2
Rv1993c
cmtA
Cd2+
Cd 2+- CmtR
–10 –35
T1 T2
Rv1993c
cmtA –10 –35
Fig 8 Transcriptional regulation of the cmtR–1993c–cmtA operon
by CmtR CmtR (oval) represses transcription of the cmtR–1993c–
cmtA operon by binding to multiple sites overlapping and
down-stream of the T1 cmtR and T2 cmtR promoters, respectively Cd(II)
(black circles) acts as an inducer, binding to CmtR and releasing it
from the DNA, resulting in the de-repression of operon trans
cription.
Table 1 Plasmids used in this study KmR, kanamycin resistance.
Plasmid Description
Source or reference pPROEX-HTb E coli expression vector
(N-terminal histidine tag)
Invitrogen pGEMT-Easy E coli cloning vector Promega pFPV27 E coli–mycobacteria
shuttle plasmid containing
a promoterless GFP gene, Km R
[32]
pPRO-CmtR cmtR coding region in
pPROEX-HTb to overexpress CmtR with a N-terminal histidine tag
This study
pCmtR pFPV27 containing the
156 bp Rv1994c–Rv1995 intergenic region promoter (P6–P3 fragment) upstream
of GFP
This study
Trang 10sonicate was centrifuged at 12 000 g for 30 min and the
supernatant was applied to a nickel-nitrilotriacetic acid
column Recombinant His-CmtR protein (approximately
16.0 kDa) was eluted in buffer A containing 250 mm
imid-azole The histidine tag was removed using rTEV protease
(Invitrogen) according to the manufacturer’s protocol to
yield a protein of approximately 12.5 kDa, referred to as
CmtR The purified His-CmtR and CmtR proteins were
dialyzed against 50 mm Tris (pH 8.0), 50 mm NaCl and
50% glycerol, and stored at)20 C
SDS–PAGE, western blotting and formaldehyde
crosslinking
Purified His-CmtR or CmtR was boiled for 5 min at
100C in SDS sample buffer (300 mm Tris ⁄ HCl pH 6.8,
12% SDS, 60% glycerol, 0.6% bromophenol blue) in the
absence and presence of 7.5% b-mercaptoethanol or
various concentrations of dithiothreitol The samples were
resolved by 12% SDS–PAGE The bands were transferred
to nitrocellulose membrane at 0.8 mAÆcm2 )1for 2 h using a
semidry blotting apparatus (Bio-Rad, Hercules, CA, USA) The membrane was probed with horseradish peroxidase-conjugated anti-His serum (Qiagen, Valencia, CA, USA) or polyclonal antibody against purified recombinant M tuber-culosisCmtR protein raised in rabbit and processed accord-ing to the manufacturer’s protocol (Qiagen) or as described previously [30] A crosslinking experiment with purified His-CmtR was performed in the presence of 0.1% formal-dehyde in standard phosphate-buffered saline [29] for
30 min at 25C Crosslinking was terminated by the addi-tion of SDS sample buffer (with 7.5% b-mercaptoethanol), and the products were resolved by 10% SDS–PAGE before transfer to nitrocellulose membrane as described above The membrane was probed with horseradish peroxidase-conjugated anti-His serum (Qiagen) and developed using 3’,3’-diaminobenzidine
Electromobility shift assay and DNase I footprinting
EMSA and DNase I footprinting were performed with purified CmtR (His tag removed) For EMSA, radiolabeled DNA fragments were generated by PCR using appropriate primers (Table 2 and Fig 5A), one of which was end-labeled using c-32P ATP (approximately 3000 CiÆmmol)1, Board of Radiation and Isotope Technology, Hyderabad, India) Binding of CmtR was performed in a 20 lL reac-tion where the protein was first incubated with metal ions for 10 min at room temperature (when required) and then with 32P-labeled DNA (approximately 2 ng, approximately
15 000 cpm) or with double-stranded oligonucleotides for
30 min on ice in binding buffer [25 mm Tris⁄ HCl pH 8.0,
6 mm MgCl2, 5% glycerol, 0.02 mm dithiothreitol and 1 lg
of poly(dI-dC)] The reaction was electrophoresed on a 5% non-denaturing gel at 120 V (constant) in 0.5· Tris ⁄ borate buffer at 4C after pre-running the gel for 30 min under similar conditions The gel was dried and analyzed by phosphor imaging using Quantity One software (Bio-Rad)
To make double-stranded oligonucleotides, single-stranded oligonucleotides (Table 2) were annealed by incubating at
95C for 3 min in buffer containing 10 mm Tris ⁄ HCl and
100 mm NaCl, and allowed to cool slowly to 4C The DNA was visualized by ethidium bromide staining
The DNase I footprinting assay was performed as described previously [26] The binding and running buffers used were the same as in EMSA DNA–protein interac-tion was performed as described above with approximately
150 000 cpm of labeled DNA, in a reaction volume of
50 lL DNase I treatment with 0.2 units was performed for 3 min at 22C in the presence of 50 lL cofactor solu-tion (2.5 MgCl2 and 5 mm CaCl2), and the reaction was stopped by the addition of 90 lL stop solution (200 mm NaCl, 30 mm EDTA, 1% SDS and 66 lgÆmL)1 yeast tRNA) The reaction products were phenol⁄ chloroform-extracted, ethanol-precipitated, washed with 70% ethanol
Table 2 List of primers ⁄ oligonucleotides used in the study.
Primers Sequence (5¢- to 3¢) Experiment
P1 GTACTATTGGATCCATGCTGACG CmtR protein
expression P2 GTCCTGTAAGCTTAAGTCGTGTC CmtR protein
expression P3 TTCCCGCATCTCACACGTCA Reporter assay
P6 GTCACACCTTTCGTCGCAGC Reporter assay,
EMSA, DNase
I footprinting P8 TGTTATACCAGTATATGGTGTACTA EMSA
94RTf CTCGGCCTCAACTACAGTCGT Reverse transcription
94RTr ACAGGTAGCTGAGCAGCAGAC Reverse transcription
1994intR CAGCTAGCTGGCCGGGATAGC EMSA, DNase I
footprinting, TSP mapping
oligonucleotides for site 1 P1R CCATAGCAGATATGATCGGC
oligonucleotides for site 2 P2R AGCAAGAGCTGAATTGTACAT
oligonucleotides for site 3 P3R CCATAT ACTGGTATAACAGC
oligonucleotides for site 4 P4R CATAGATCAAATAGTACACCA
H1 CGAGTCGACCGGAGGACCTTT
GGCCCTGCGTCGACCGA
EMSA, oligonucleotides used in control experiment H2 TCGGTCGACGCAGGGCCAAAG
GTCCTCCGGTCGACTCG