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J. Biol. Chem., Vol. 278, Issue 36, 33763-33773, September 5, 2003
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From the
Unité de Génétique
Mycobactérienne, Institut Pasteur, 25, Rue du Dr. Roux, 75724 Paris
cedex 15 and the ¶Unité d'Enzymologie et
Cinétique Structurale, CNRS/Ecole Normale Supérieure de Cachan,
61, Avenue du President Wilson, 94230 Cachan, France
Received for publication, June 6, 2003 , and in revised form, June 20, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The genome of M. tuberculosis contains six predicted transcriptional regulators belonging to the AraC family (43): Rv1317c (alkA (44)), Rv1395, Rv1931c, Rv3082c (virS (45, 46)), Rv3736, and Rv3833. Members of the AraC family mainly act as activators and only rarely as repressors (47–49). The proteins can be divided into two domains. The N-terminal region, which varies largely among members, is recognized by the effector molecules and is responsible for dimerization. The C-terminal region recognizes and binds the target DNA by two helix-turn-helix motifs that are conserved among organisms and represents the "signature" of the family. Rv1395 is a potential transcriptional regulator of the AraC family. It was identified by signature-tagged mutagenesis in M. tuberculosis and the Rv1395 insertional mutant was shown to be attenuated in the lungs in the mouse model of tuberculosis (50). Rv1395 can be divided into two domains, and it has two predicted helix-turn-helix motifs in the C-terminal domain, recognized as members of the AraC family (43). The aim of this study was to identify Rv1395 target genes and to characterize its regulatory activity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Bacterial Strains—M. smegmatis mc2155 and M.
tuberculosis MT103 and H37Rv were grown in Middlebrook 7H9 medium (BD
Biosciences) supplemented with albumin/dextrose/catalase (from BD Biosciences)
and 0.05% Tween 80, at 37 °C. The M. tuberculosis
Rv1395– strain is the 2B26 strain isolated by Camacho et
al. (50). E.
coli DH5
and BL21 were grown at 37 °C in L-broth. When
required, antibiotics were added at the following concentrations: ampicillin,
100 µg ml–1; kanamycin, 20 µg
ml–1; hygromycin, 200 µg
ml–1 (for E. coli) or 50 µg
ml–1 (for mycobacteria). Competent E.
coli cells were prepared by the CaCl2 method and transformed
according to Sambrook et al.
(55). Competent M.
smegmatis and M. tuberculosis cells were prepared and
transformed by electroporation as described by Guilhot et al.
(56).
In Vitro Binding Assay—Crude extracts were prepared by
resuspending pellets from 2- to 3-week-old M. tuberculosis cultures
in 500 µl of lysis buffer (10 mM Tris-HCl buffer, pH 8.0, 1
mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1
mM phenylmethylsulfonyl fluoride) and breaking the bacteria with
500 µl of 0.1-mm glass beads. Soluble proteins were separated from cell
debris by centrifugation and quantified by use of an ESL kit (Roche Applied
Science). Radioactive probes were prepared by phosphorylating 10 pmol of one
of the primers with [
-32P]ATP (3000 µCi/mM)
and T4 polynucleotide kinase (BioLabs) and adding the labeled primer to PCR
reactions. Binding assays were carried out in 10 µl of binding buffer (43
mM Tris acetate, pH 8.0, 30 mM potassium acetate, 8
mM magnesium acetate, 27 mM ammonium acetate, 1
mM dithiothreitol, 80 mM KCl, 10% glycerol, 4%
polyethylene glycol 8000, 100 µg/ml bovine serum albumin, 100 ng/ml
poly(dI-dC)) at 30 °C for 30 min and run in 10% polyacrylamide gels. The
intensity of the bands was quantified with ImageQuant (Amersham
Biosciences).
Purification of Rv1395Cter—All PCRs were carried out with
the Pfu Turbo polymerase from Stratagene and with genomic DNA from
M. tuberculosis as a template. The C-terminal region of Rv1395 was
amplified using primers TR9c
(5'-CATGCCATGGCACAATGCGACGTGCTGATG-3') and Tr12bis
(5'-GGAATCCATATGCTAGTGGTGATGGTGGTGGTGCCCGCGGCGCGAATATTCGCT-3'),
cloned into the NcoI/NdeI sites of pET14b (Novagen), and
sequenced. The resulting plasmid was named pET 9c-12b. E. coli BL21
was transformed with pET 9c-12b and induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside at
A600 = 0.5 for 5 h. Bacteria were harvested by
centrifugation, washed, resuspended in lysis buffer (10% glycerol, 0.5
M NaCl, 20 mM Tris-HCl buffer, pH 8.0), and broken by
sonication. The resulting pellet (insoluble fraction) was resuspended
overnight at 4 °C in urea buffer (8 M urea, 0.5 M
NaCl, 20 mM Tris-HCl buffer, pH 8.0) at pH 8.5. After
centrifugation at 8000 x g, the supernatant was loaded in an
equilibrated nickel-nitrilotriacetic acid-agarose (Qiagen) column. The column
was washed with urea buffer (8 M urea, 0.5 M NaCl, 20
mM Tris-HCl buffer, pH 8.0) at pH 6.6, and the protein eluted with
urea buffer (8 M urea, 0.5 M NaCl, 20 mM
Tris-HCl buffer, pH 8.0) at pH 4.5. Fractions were first analyzed on gels,
then gathered and dialyzed in the presence of 0.05% salmon sperm DNA against
phosphate-buffered saline
(57). The protein was then
centrifuged at 12,000 x g, and the soluble fraction was
analyzed: after dialysis, the fractions that had been eluted at pH 
5.5
appeared as a single band in a Coomassie Blue-stained polyacrylamide gel.
These fractions were divided into aliquots and stored at –80 °C.
Preparation of Anti-Rv1395 Antibodies—Polyclonal anti-Rv1395 antibodies were raised by immunizing rabbits with recombinant Rv1395. The full-length protein was obtained by cloning the PCR product obtained with primers TR9 (5'-CATGCCATGGGACATCTACCGCCTCCGGCC-3') and TR12bis into pET14b (Novagen). The protein was purified as described for the C terminus. The purified protein was highly degraded and showed poor binding capacity. New Zealand rabbits were immunized with 200 µg of recombinant Rv1395; two boosters (200 µg) were given at 2-week intervals. Blood was collected, incubated for1hat 37 °C and overnight at 4 °C. Sera were recuperated by centrifuging the blood at 3000 x g for 20 min.
DNase I Footprinting—The binding assay was performed as described above. DNase I (40 ng) was then added, and the tubes were incubated at 30 °C for 2 min. The reaction was stopped by adding 5 µl of formamide loading buffer. Sequencing was performed using the T7 DNA Polymerase Sequencing Kit from USB. Samples were run in a urea-denaturing 7.5% polyacrylamide gel.
Primer Extension—RNA was extracted from late
logarithmic-phase cultures of recombinant M. smegmatis and M.
tuberculosis H37Rv as follows: culture pellets were resuspended in 1 ml
of TRIzol (Invitrogen) and broken with 500 µl of 0.1-mm glass beads for 1
min. Then, 200 µl of chloroform:isoamyl alcohol (49:1) (Ready Red from
Appligene) was added, and the mixture was incubated for 3 min at 23 °C.
Tubes were centrifuged at 15,000 x g for 15 min, and the
supernatant was precipitated with 500 µl of isopropanol at 23 °C.
Pellets were washed with 75% ethanol and resuspended in 50 µl of diethyl
pyrocarbonate-treated water (Ambion). The RNA preparations were treated with
DNase with the DNA-free kit from Ambion. For primer extension analysis,
2–5 µgof RNA was mixed with 2 pmol of radioactive primer TR+1
(5'-AGCACCCGGGTCGCATACACC-3') for Rv1395, primer Cyt+1
(5'-TCATCGTCCAGGTGCTCATCC-3') for Rv1394c, primer Gal+1
(5'-GAAGCTTCCGATTCGTAGAGC-3') for -galactosidase fusions,
and reverse-transcribed with Expand Reverse Transcriptase (Roche Applied
Science) following the manufacturer's instructions. Samples were run in a
urea-denaturing 7.5% polyacrylamide gel.
Integration of Rv1395 into the Chromosome—Rv1395 and its promoter region were PCR-amplified with primers TR1 (5'-GGACTAGTTGGTGCATGTCGAACTGCGC-3') and TR2 (5'-CCACTAGTGCCTAACCCGACCAAACCGAC-3'). The fragment was digested with SpeI and cloned into the SpeI site of the integrative vector pOIP 23 (58). The resulting plasmid pOIP 1-2 was electroporated into M. tuberculosis strains MT103 Rv1395– and H37Rv and into M. smegmatis.
Construction of the -Galactosidase
Fusions—The region between Rv1395 and Rv1394c was PCR-amplified
with primers TRgal1
(5'-CCGGATCCTGGGTGGTGGCGGTGGCCAT-3') and TRgal2
(5'-CCGGATCCGGAGGCGGTAGATGTCCCAC-3'), digested with
BamHI, and cloned into the BamHI site of pJEM15
(59). Sequencing revealed the
orientation of the region respective to the -galactosidase gene, giving
rise to plasmids pCyt and pTR. Plasmid pTR-L was obtained by cloning the
BamHI-digested PCR product obtained with primers TRgal2-TR1 into
ScaI/BamHI-digested pJEM15. Plasmid pCyt-L was obtained by
cloning the ScaI/BamHI-digested PCR product obtained with
primers TRgal1-TRHrSca
(5'-AAAAGTACTGGTAAACGTTGGGAAAAGCCC-3') into
ScaI/BamHI-digested pJEM15. The plasmids were sequenced and
used to transform M. smegmatis and M. smegmatis:Rv1395, M.
tuberculosis H37Rv wild type, and M. tuberculosis H37Rv (pOIP
1-2).
-Galactosidase Assay—Recombinant M. smegmatis
and M. tuberculosis were grown to late-logarithmic phase, harvested
by centrifugation, resuspended in 500 µl of 0.1 M sodium
phosphate buffer, pH 7.0, and broken with 0.1-mm glass beads. One hundred
microliters of the supernatant fraction was mixed with 600 µl of 4
mM O-nitrophenyl -D-galactopyranoside,
0.1 M sodium phosphate buffer, pH 7.0, and optical densities were
read at 420 nm. -Galactosidase activity was measured as the increase in
A420 x 1000 per mg of protein per minute. Results
are the average of at least three independent experiments.
Site-directed Mutagenesis—Fragments containing the mutated
sites were constructed as follows. The PCR products of primers TR1Bam
(5'-CCGGATCCTTGGTGCATGTCGAACTGCGC-3') and MutLow
(5'-CGGAATTCGACTCTGCCTCGGGCCAGTCGGCGTCATCC-3'), TR1Bam
and EcoLow (5'-CGGAATTCTTGTCCCATAGTCTCATTTCGGGC-3'),
TRn (5'-CGCGATGGTAAGCAGTTCGCG-3') and MutHigh
(5'-CGGAATTCACAGACCCGAAGTAGCCTCGTGGGACATCTACCG-3'), and
TRn and EcoHigh
(5'-CGGAATTCCAAAGTGTTGTGAGTTTAGGACAG-3') were named
MutLow, EcoLow, MutHigh, and EcoHigh, respectively. They were all digested
with EcoRI, MutLow was ligated with EcoHigh, and MutHigh was ligated
with EcoLow. The ligations were amplified with primers TR1Bam and TRgal2, and
the resulting products were gel-purified and named TRL-MutL and TRL-MutH,
respectively. These fragments were subsequently amplified with primers TRgal1
and TRgal2 to give rise to PrGal-MutL and PrGal-MutH, respectively. PrGal-MutL
and PrGal-MutH were phosphorylated with [-32P]ATP and used
for the binding assay, as previously described. TRL-MutL, TRL-MutH,
PrGal-MutL, and PrGal-MutH were digested with BamHI and cloned into a
BamHI-digested pJEM15. The resulting plasmids were sequenced for
correct orientation and named pTRL-MutL, pTRL-MutH, pCyt-MutL, and pCyt-MutH,
respectively. M. smegmatis and M. smegmatis:Rv1395 were
transformed with these plasmids.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We compared the region encompassing Rv1395 in M. tuberculosis and in the other mycobacteria for which the genome sequence is available. The genomic region of M. tuberculosis H37Rv contained, on each side of Rv1395, two genes widely conserved among prokaryotes (54, 62). These were metK (COG 0657), an S-adenosyl methionine synthetase, and lipH (COG 0192), a member of the lipase-esterase family (Fig. 1). In M. tuberculosis, the region comprised between metK (Rv1392) and lipH (Rv1399c) contains six genes possibly encoding a monooxygenase (Rv1393c), a member of the cytochrome P450 family (Rv1394c), Rv1395, a member of the PGRS family (Rv1396c), an unknown product (Rv1397c), and a putative regulator of the CopG family (Rv1398c) (Fig. 1). The two genes encoding the cytochrome P450 and the monooxygenase are probably organized in an operon, because the stop codon of the former overlaps the initiation codon of the latter. We first explored the Rv1395 region of the other members of the M. tuberculosis complex: in M. tuberculosis CDC1551 the region was identical except for a frameshift at the beginning of the PGRS gene; the locus was also conserved in Mycobacterium microti and in M. bovis, although there was a 374-bp deletion in the middle of the PGRS gene of the latter. The metK-lipH region was also present in the vaccine strain M. bovis BCG, but lipH, Rv1395, and the PGRS genes had frameshift mutations at amino acids 84, 329, and 87, respectively. Thus, the Rv1395 region appears to have been conserved throughout the evolution in the mycobacteria of the M. tuberculosis complex. We then examined the locus in other mycobacterial species. In M. leprae, described as an extreme case of reductive genomic evolution (63), the region between metK and lipH is completely devoid of any ORF and limited to 663 bp (Fig. 1). In M. avium, the region encodes four potential products: two monooxygenases (one of which is similar to Rv1393c), one protein of unknown function similar to a Streptomyces coelicolor putative protein annotated as SCL24.07, and a potential regulator of the AsnC family (Fig. 1). However, the potential regulator of the AsnC family and Rv1395 do not share any similarity. The region of M. marinum is similar to that of M. avium, but the central region composed of the AsnC regulator and the first monooxygenase is absent (Fig. 1). In M. smegmatis, the region encodes three potential genes (Fig. 1) with similarity with the E. coli genes encoding the D-methionine ABC transporter (64), which have the same tandem organization in E. coli. No Rv1395 homologues were found elsewhere in the genomes of M. leprae, M. avium, M. marinum, and M. smegmatis. This analysis illustrates the extreme plasticity of the metK-lipH locus in mycobacteria and demonstrates that Rv1395 is a potential regulator unique to the M. tuberculosis complex.
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Rv1395 Binds to the Intergenic Region of Rv1395-Rv1394c by Its
C-terminal Moiety
To investigate the DNA-binding properties of Rv1395, a gel mobility assay
was developed using crude extracts of M. tuberculosis. Because
regulators often act on genes located in the vicinity of their own gene, a
binding assay was performed on each of the three intergenic regions unique to
the M. tuberculosis complex in the metK-lipH locus
(Fig. 1). This experiment was
done using protein extracts of M. tuberculosis wild type and
Rv1395–. No band shift was observed with the putative
promoter regions of the PGRS or the CopG regulator (data not shown). On the
contrary, a clear band shift was observed with the region between
Rv1395 and Rv1394c with the extract of wild type M.
tuberculosis but not with the Rv1395– extract
(Fig. 2A). Two shifted
complexes could be seen on the gel. Moreover, these complexes were also
observed with extracts of the Rv1395– strain complemented
with an intact copy of Rv1395
(Fig. 2A), although
the low mobility complex was more abundant than the high mobility complex. The
amount of shifted complexes correlated well with the amount of Rv1395 present
in the extracts (Fig.
2B). The overexpression of Rv1395 observed in
the complemented strain is probably due to a cryptic promoter present in the
integrative plasmid used for complementation
(58). The complexes were
specific, i.e. they were lost following the addition of an excess of
cold probe and conserved in the presence of an excess of nonspecific DNA
(Fig. 2C). The two
complexes were lost at different concentrations of the cold probe, indicating
that the bound proteins had a different affinity for the DNA. This result
indicates that Rv1395 itself or another molecule regulated by Rv1395 is
responsible for the observed complexes.
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To test whether Rv1395 interacted directly with the region between Rv1395 and Rv1394c, we attempted to purify Rv1395 from an overproducing E. coli strain. However, proteins of the AraC family are often insoluble when overproduced in E. coli. To circumvent this problem, we reasoned that a single domain of Rv1395 might remain active in solution more easily. The helix-turn-helix-containing C-terminal domain was thus chosen for purification. This domain (Rv1395Cter, amino acids 229–344) was His-tagged, overproduced in E. coli, and purified from inclusion bodies in denaturing conditions. Rv1395Cter was then resolubilized and kept in solution in the presence of nonspecific DNA using the method of Egan et al. (57). The soluble protein was tested in a binding assay. Rv1395Cter efficiently bound to the Rv1395-Rv1394c intergenic region, and a shifted product was obtained with as little as 1 nM protein (Fig. 2D). Similarly to the crude extract, two shifted products were observed with the purified Rv1395Cter. Quantification of these data (Fig. 2E) resulted in a Kd = 5 nM for the high mobility, high affinity complex and a Kd = 125 nM for the low mobility, low affinity complex. These complexes were specific, i.e. lost following the addition of an excess of cold probe and conserved in the presence of an excess of nonspecific DNA (data not shown). The second less mobile complex suggests that a second Rv1395Cter molecule binds either to another site in the DNA or to the bound protein. The complexes observed with Rv1395Cter were more mobile than those obtained with the crude extracts. This may be explained by the difference in size between the full-length protein present in the crude extracts and the purified C-terminal domain. In addition, another molecule present in the extracts may bind to the DNA. These data demonstrate that Rv1395 binds directly and specifically to the region between Rv1395 and Rv1394c by its C-terminal domain.
Identification of the Binding Sites of Rv1395
The very high affinity of Rv1395 for its target DNA enabled us to
characterize its binding site. We carried out a DNase I protection assay using
the purified Rv1395Cter or a crude extract of M. tuberculosis. When
the intergenic region was incubated with various concentrations of purified
Rv1395Cter and then treated with DNase I, two regions were protected
(Fig. 3A). A first
protected region of 26 bp (5'-caaagtgttgtgagtttaggacagcc-3')
appeared with the same affinity as the first, high affinity complex in the
gel-shift assay. A second region of 16 bp (5'-aaatgagactatggga-3')
was protected at higher protein concentrations and corresponded to the second,
low affinity shifted complex in the binding assay. This result shows that
Rv1395 binds to two sites in the Rv1395-Rv1394c intergenic region
with different affinities. The first region has two hypersensitive sites,
whereas the second region has only one
(Fig. 3A).
Hypersensitive sites indicate increased exposure of DNA to DNase, and they are
usually due to the bending of the DNA caused by the bound molecule
(65).
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To test whether the protected regions defined using the purified C-terminal domain were also protected by the entire protein, a DNase I-footprinting experiment was carried out using crude extracts of M. tuberculosis. We used the complemented strain that overexpresses Rv1395, because the signal with the wild type strain was too weak. The protected region obtained with the crude extract was similar to that obtained with the C-terminal domain of Rv1395 but longer (Fig. 3B). Indeed, a further 8-bp region (5'-ctggcccg-3') was protected upstream from the second region. This could be due to the fact that the entire protein covers a wider region or, alternatively, that another molecule present in the extract binds to this region. However, this region was not protected with extracts from the Rv1395 mutant strain (data not shown). Furthermore, the first protected region has only one of the two hypersensitive sites observed with the purified protein (Fig. 3B). Again, this could be due to the different length of Rv1395 in the two assays or to the interaction with another molecule that modifies the DNA-bending properties of Rv1395. This result confirms that Rv1395 binds essentially to the same regions both when it is purified in denaturing conditions from an overproducing E. coli strain and when it is found in extracts from mycobacteria.
Identification of the Transcriptional Start Points
As shown above, Rv1395 efficiently binds to the region between
Rv1395 and Rv1394c. Because the binding sites are close to
the predicted start codons of both the cytochrome P450 and Rv1395
(Fig. 4C), Rv1395
probably binds within both promoter regions and affects their transcription
level. To define the promoter regions, we used primer extension analysis to
locate the transcriptional start points of Rv1395 and
Rv1394c in M. tuberculosis
(Fig. 4, A and
B, respectively). Both genes were transcribed from two
promoters, and P1 indicates the promoter that is closer to the
predicted ATG. Curiously, P1 coincides with the predicted start
codon in both genes (Fig.
4C), a phenomenon that has already been observed in
M. tuberculosis (22,
40,
59), in M. leprae
(66) and in other
Actinomycetales (67). However,
it is possible that the real start codon is located further downstream.
Indeed, there are other amino acids that could act as start codons: two
methionines are located at positions 17 and 22 with respect to the predicted
ATG in Rv1394c and other six valines are found among the first 34 predicted
amino acids in Rv1395. It should be noted that the region of similarity
between Rv1395 and P. aeruginosa proteins PA5324 and PA5032 starts at
amino acid 15 and the 14th amino acid is a valine, suggesting that this latter
could be the real start codon.
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Characterization of the Transcriptional Properties of Rv1395
Because M. smegmatis is phylogenetically close to M.
tuberculosis and devoid of the Rv1395 region, this species was
used as a surrogate host to study the transcriptional properties of Rv1395.
The gene encoding Rv1395 was integrated into the M. smegmatis
chromosome using the same integrative plasmid used to complement the M.
tuberculosis mutant strain. Crude extracts were prepared from wild type
M. smegmatis and M. smegmatis:Rv1395 and tested in a binding
assay with the intergenic region as a probe: two shifted complexes were
observed with extracts of the recombinant M. smegmatis but not with
extracts of the wild type (data not shown). The complexes were the same sizes
as those observed with M. tuberculosis extracts. This result proves
that Rv1395 is expressed in M. smegmatis:Rv1395 and that it retains
its DNA-binding activity. M. smegmatis:Rv1395 and wild type M.
smegmatis were then used to study the impact of Rv1395 binding on the
expression level of both Rv1394 and Rv1395.
Rv1395 Is an Activator of the Cytochrome P450 cyp132 Gene—We
studied the effect of Rv1395 on the expression of the cytochrome P450 gene by
constructing transcriptional fusions with the -galactosidase reporter
gene. Two fusions were constructed (Fig.
5I, panel a): a short one including only the
P1Cyt region (pCyt) and a long one including both
P1Cyt and P2Cyt (pCyt-L). The
long fusion was used to investigate whether Rv1395 could activate
P2Cyt from a downstream position as has been described for
Rns (68) and other regulators
of the AraC family (69). In
this regard, Rv1395 did not bind upstream from P2Cyt nor
downstream from P1Cyt (data not shown).
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Wild type M. smegmatis and M. smegmatis:Rv1395 were
transformed with pCyt and pCyt-L, and the amount of -galactosidase
produced was measured. As seen in Fig.
5I (panel b), the two promoters had a very
different basic activity in wild type M. smegmatis, with
P2Cyt being 16 times stronger than
P1Cyt. However, the activity of P1Cyt
(73 ± 17 units) was important, because it was higher than that of the
empty vector (1 ± 1 units) (data not shown). Thus, both M.
tuberculosis promoters are recognized by the M. smegmatis
transcriptional machinery. When Rv1395 was added in trans, the
activity of P1Cyt increased of a factor 17 in M.
smegmatis:Rv1395 (pCyt) (Fig.
5I, panel b). An increase in the activity was
also observed with the pCyt-L construct in the presence of Rv1395, but this
increase was mainly due to the induction of P1Cyt. This
indicates that Rv1395 induces the expression of the cytochrome gene by
activating P1Cyt and that it has no major effect on
P2Cyt expression. To confirm this observation, we used
primer extension to measure the amount of RNA transcripts in M.
smegmatis (pCyt-L) and M. smegmatis:Rv1395
(pCyt-L) (Fig.
5I, panel c): P1Cyt was
clearly induced in the presence of Rv1395.
Rv1395 Represses Its Own Expression—To study the effect of
Rv1395 on its own expression, two fusions were made
(Fig. 5II, panel
a): a short one containing only P1Rv1395
(pTR, where TR stands for transcriptional regulator) and a long one
comprising both P1Rv1395 and P2Rv1395
(pTR-L). Wild type M. smegmatis and M.
smegmatis:Rv1395 were transformed with the two constructs and the
-galactosidase activity measured. In wild type M. smegmatis,
the promoter activity of P2Rv1395 was 15 times stronger
than that of P1Rv1395
(Fig. 5II, panel
b). In the recombinant M. smegmatis:Rv1395, both promoters were
repressed by Rv1395. P1Rv1395 activity was repressed by
only 20%, whereas P2Rv1395 activity was repressed by 74%
(Fig. 5II, panel
b). Thus, Rv1395 is a repressor of its own expression, a behavior that is
common among transcriptional regulators.
Rv1395 Activity in M. tuberculosis—To confirm that the situation is the same in M. tuberculosis as in M. smegmatis, the same fusions were electroporated into wild type M. tuberculosis and into an Rv1395-overexpressing strain to compare the effect of basic and high Rv1395 expression. Rv1395 was overexpressed in M. tuberculosis by means of the same integrative plasmid previously used in M. smegmatis:Rv1395. The results were very similar to those observed in M. smegmatis (Fig. 5III). Both P2 promoters had higher activity than their respective P1 promoters and P1Cyt activity increased following Rv1395 overexpression (Fig. 5III, panel a), whereas P1Rv1395 and P2Rv1395 activities were reduced by 43 and 74%, respectively (Fig. 5III, panel b). Thus, the regulation properties of Rv1395 in M. tuberculosis are the same as those observed in M. smegmatis.
The Binding to the High and Low Affinity Sites Is Cooperative and
Necessary for the Full Activity of Rv1395
To define the binding properties of the two binding sites recognized by
Rv1395 and their role in the induction/repression activity of Rv1395, each
site was mutated separately. As the specific bases within the protected
regions recognized by Rv1395 were not known, we decided to mutate 19 bp in the
high affinity site (giving rise to mutH) and 15 bp in the low affinity site
(named mutL) (Fig.
6A). We simply replaced purines by purines and
pyrimidines by pyrimidines. The two mutated regions, mutL and mutH, were first
analyzed by binding assay with crude extracts: mutL showed a single shifted
band similar to the lower complex observed with the wild type region, whereas
mutH did not show any shifted complexes at all, even with the extracts from
the Rv1395-overexpressing strain
(Fig. 6B). This
suggests that Rv1395 binds to the high affinity site independently of the
presence of the low affinity site, whereas the binding to the low affinity
site is co-operative and requires the presence of the other molecule bound to
the high affinity site. We also analyzed the two regions by binding assay with
the Rv1395Cter. In both cases only one shifted complex was obtained (data not
shown). The binding affinity for mutL was the same as for the "wild
type" fragment, with a Kd of 5 nM (data
not shown). On the contrary, in mutH, the Kd for the low
affinity site increased by one log (data not shown). This confirms that, in
the absence of the high affinity site, Rv1395 binds very poorly to the low
affinity site. This cooperation could be explained either by the direct
interaction between the proteins bound to the two sites or by the fact that
the molecule bound to the first site bends the DNA so to increase the affinity
of the second site. Indeed, the cooperativity between the two sites may be
higher in the presence of the N-terminal domain, because this region is often
responsible for the dimerization of transcription factors.
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These mutations were also inserted into the vectors pCyt and pTR-L to study the effect of the mutations on the regulatory activity of Rv1395. The resulting plasmids pCyt-MutL, pCyt-MutH, pTRL-MutL, and pTRL-MutH were electroporated into M. smegmatis and M. smegmatis:Rv1395. In the case of the cytochrome P450, the mutations did not alter the basal promoter activity of P1Cyt, but they significantly modified the induction by Rv1395 (Fig. 6C). Interestingly, in the Cyt-MutL fusion, where Rv1395 binds only to the high affinity site, the cytochrome was induced just by a factor two (Fig. 6C), whereas in the "wild type" construct the cytochrome was induced 17-fold in the presence of the regulator (see Fig. 5I, panel b). This decrease in the induction level indicates that both sites are required for cytochrome activation and that, if Rv1395 binds solely to the high affinity site, it is not able to produce an optimal induction. On the other hand, no induction was observed in the Cyt-MutH fusion (Fig. 6C). This can easily be explained by the fact that, as shown previously by the binding assay with crude extracts, Rv1395 does not bind to the low affinity site when the high affinity site is mutated and so no Rv1395 molecule bound to the mutH region. The same reasoning may also explain the results in M. smegmatis:Rv1395 pTRL-MutH (Fig. 6D), where the fusion was not repressed by the presence of Rv1395, probably because Rv1395 did not bind to the mutated DNA. It should be noted that the mutH mutation did not modify P2Rv1395 basal activity. On the contrary, the mutL mutation reduced the basal activity of P2Rv1395 by more than 10 times (Fig. 6D). This result was quite unexpected, because this last mutation is located several base pairs downstream from the transcriptional start point of P2Rv1395. However, in the mutL fusion the binding of Rv1395 to the high affinity site did not repress this weaker P2Rv1395 activity. This observation confirms that both sites are required for optimal Rv1395 activity.
Study of the Recognition Motif for Rv1395
The experiments with the mutated fragments mutL and mutH showed that the
nucleotides contained in the two binding sites are necessary for recognition
by Rv1395. To identify the recognition motif of Rv1395, the sequences of the
two sites (shown in Fig.
4C) were searched for conserved patterns. Relative to the
hypersensitive sites, a conserved TGTGA sequence was observed repeated
symmetrically in the first region (agtgt/Tgtga) and with one mismatch in the
second region (Tgaga), where the capital T is the hypersensitive
site. However, regulators usually recognize directed or inverted repeats and
not symmetrical elements. Another common motif between the two sites was
TGAG-n-GGACA, where n = 4 in the high affinity region and
n = 6 in the low affinity region. To determine whether this motif was
sufficient for Rv1395 binding and could allow the direct identification of
other putative targets, we sought the TGAG-n-GGACA motif and its
complement sequence TGTCC-n-CTCA in the whole M.
tuberculosis genome in the 200-bp region upstream from all the annotated
ORFs. Interestingly, seven genes presented the TGAG-n-GGACA motif and
five genes presented its complementary sequence between 27 and 179 bp upstream
from their predicted ATG (Table
I). However, no genes contained multiple copies of the repeat in
the promoter region. Three genes were chosen for further analysis:
Rv2728c, Rv2781c, and Rv2322c, with n = 4, 5, and 6
respectively (Table I).
Different values of "n" were chosen to investigate
whether the number of bases between the two parts of the motif could explain
the different affinity of Rv1395 for the two sites. Furthermore,
Rv2322c encodes for an ornithine aminotransferase and Rv1395 is
similar to OruR, a transcriptional regulator involved in ornithine metabolism
in P. aeruginosa. The promoter regions of the three genes were
amplified and tested in binding assays with recombinant Rv1395Cter or with
crude extracts. However, no binding was observed in any case (data not shown).
This suggests that the TGAG-n-GGACA motif is not sufficient per
se for recognition by Rv1395 and that other adjacent bases are also
required for the high specificity of Rv1395 for the Rv1394c-Rv1395
region.
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DISCUSSION |
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Here we show that Rv1395 is an activator of the cytochrome P450 gene
Rv1394c and that it acts on its P1 promoter
(Fig. 7). Rv1395 binds to two
sites relative to P1Cyt: the high affinity site is located
upstream from the –35 box, around position –62.5, whereas the low
affinity site is centered around position –41.5 and partially overlaps
the –35 region (Fig. 7).
These are the positions typically occupied by regulators of class I and class
II promoters, respectively
(77–79).
In class I promoters, the activator molecule binds upstream from the –35
region, typically at –62, where it makes contacts between its
"activating region" (AR1) and the CTD domain of the RNA
polymerase (79). This
interaction increases the binding constant of the RNA polymerase. In class II
promoters, the activator molecule binds around –42 and overlaps the
–35 region, where it can make multiple interactions with the CTD
domain, the NTD domain, and the region 4 of the sigma factor via its
AR1, AR2, and AR3 regions, respectively
(79). These contacts increase
the constant of isomerization of the closed complex to an open complex. Both
class I and class II behaviors have been described for the members of the AraC
family (49). Combinations of
class I and class II promoters give rise to class III promoters, which require
multiple activators for their full induction
(79). As binding of Rv1395 to
both sites is required for the optimal activation of the cytochrome, the
P1Cyt promoter appears to be a class III promoter and,
consequently, Rv1395 a class III activator. Given these observations and the
positions of the two sites relative to the P1Cyt promoter,
it is tempting to speculate the following model: Rv1395 binds first to the
high affinity site, around –62.5, from where either it directly recruits
another Rv1395 molecule or it bends the DNA to increase the affinity of the
second site (the binding is cooperative). As a consequence, a second molecule
of Rv1395 binds to the low affinity site at –41.5. The two bound
molecules can then interact with the RNA polymerase holoenzyme and induce the
optimal transcription of the cytochrome. In support of this model, we showed
that, if Rv1395 binds only to the –62.5 site, it induces transcription
poorly: in this case the RNA polymerase would interact only with one Rv1395
molecule and in class III promoters all activators are required at the same
time.
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Rv1395 induces the transcription of the cytochrome P450 Cyp132. Cytochrome P450s constitute a very large family of heme-thiolate monooxygenases, which are found in all living organisms (80). These enzymes oxidize a variety of compounds, including steroids, fatty acids, and xenobiotics (81). Twenty cytochrome P450 genes are present in the M. tuberculosis genome (12), and they are thought to play an important role in the rich lipid metabolism typical of mycobacteria. However, it is generally impossible to predict the nature of the substrates oxidized by cytochrome P450s on the basis of the amino acid sequence only. Therefore, the role of Cyp132 in M. tuberculosis metabolism remains to be defined.
We have also shown that, like most transcriptional regulators, Rv1395 represses its own transcription. Indeed, when Rv1395 binds to the two sites and recruits the RNA polymerase for transcription at P1Cyt, it prevents any further RNA polymerase binding at P1Rv1395 and at P2Rv1395 due to the steric hindrance of the RNA polymerase holoenzyme bound at P1Cyt (Fig. 7). Furthermore, because both sites are necessary for repression and one site has a low affinity, Rv1395 has to reach a certain concentration before it can occupy both sites at the same time and thus act as a repressor. This system makes it possible to regulate the amount of Rv1395 present in the cell.
On the other hand, the signals that might induce Rv1395 transcription are not known. Indeed, regulators are often regulated at the transcriptional level by other regulators. The fact that the basal transcription level of P1Rv1395 was reduced by more than ten times in the mutL mutant (Fig. 6D) suggests that there could be an activator site just downstream from the promoter (alternatively, the mutL mutation could just prevent the normal activity of the RNA polymerase).
The non-conserved domain is critical for signal recognition in members of the AraC family that are activated by the binding of an effector molecule. This is the case for AraC and arabinose (82) or UreR and urea (83) for examples. In addition to the interaction with small molecules, the non-conserved domain may establish productive contacts with the RNA polymerase holoenzyme and increase the affinity constant or the isomerization of the close complex into an open complex. Furthermore, this region is often responsible for dimerization and increases the cooperativity of the binding to adjacent sites. However, the similarity of the Rv1395 N terminus is restricted to some Pseudomonas putative proteins that have not yet been characterized and to the N-terminal domain of OruR, whose role and function are still unknown. This makes it difficult to predict the nature of the signal to which Rv1395 may respond and the role of this domain in modulating Rv1395 activity.
Thus the next step will be to investigate the regulation of Rv1395 transcription and activity and to determine the role of the cytochrome P450 Rv1394c in M. tuberculosis physiology and virulence.
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FOOTNOTES |
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Supported by the Fondation pour la Recherche Médicale.
|| To whom correspondence should be addressed (present address): Unité Inserm U570, Faculté deMédecine Necker-Enfants Malades, 156 Rue de Vaugirard, 75730 Paris cedex 15, France. Tel.: 33-1-40-61-53-71; Fax: 33-1-40-61-56-77; E-mail: jmreyrat{at}necker.fr.
1 The abbreviations used are: COG, clusters of orthologous groups; ORF, open
reading frame.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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