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Infection and Immunity, September 2005, p. 5988-5994, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5988-5994.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Both Corynebacterium diphtheriae DtxR(E175K) and Mycobacterium tuberculosis IdeR(D177K) Are Dominant Positive Repressors of IdeR-Regulated Genes in M. tuberculosis

Yukari C. Manabe,^1,2,3* Christine L. Hatem,¹ Anup K. Kesavan,¹ Justin Durack,¹ and John R. Murphy⁴

Department of Medicine, School of Medicine,¹ Departments of Molecular Microbiology and Immunology,² International Health, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland,³ Section of Biomolecular Medicine, Boston University School of Medicine, Boston, Massachusetts⁴

Received 4 January 2005/ Returned for modification 11 February 2005/ Accepted 27 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The diphtheria toxin repressor (DtxR) is an important iron-dependent transcriptional regulator of known virulence genes in Corynebacterium diphtheriae. The mycobacterial iron-dependent repressor (IdeR) is phylogenetically closely related to DtxR, with high amino acid similarity in the DNA binding and metal ion binding site domains. We have previously shown that an iron-insensitive, dominant-positive dtxR(E175K) mutant allele from Corynebacterium diphtheriae can be expressed in Mycobacterium tuberculosis and results in an attenuated phenotype in mice (Y. C. Manabe, B. J. Saviola, L. Sun, J. R. Murphy, and W. R. Bishai, Proc. Natl. Acad. Sci. USA 96:12844-12848, 1999). In this paper, we report the M. tuberculosis IdeR(D177K) strain that has the cognate point mutation. We tested four known and predicted IdeR-regulated gene promoters (mbtI, Rv2123, Rv3402c, and Rv1519) using a promoterless green fluorescent protein (GFP) construct. GFP expression from these promoters was abrogated under low-iron conditions in the presence of both IdeR(D177K) and DtxR(E175K), a result confirmed by reverse transcription-PCR. The IdeR regulon can be constitutively repressed in the presence of an integrated copy of ideR containing this point mutation. These data also suggest that mutant IdeR(D177K) has a mechanism similar to that of DtxR(E175K); iron insensitivity occurs as a result of SH3-like domain binding interactions that stabilize the intermediate form of the repressor after ancillary metal ion binding. This construct can be used to elucidate further the IdeR regulon and its virulence genes and to differentiate these from genes regulated by SirR, which does not have this domain.

Top Abstract Introduction Materials and Methods Results Discussion References

 
DtxR (diphtheria toxin repressor) is an iron-dependent repressor in Corynebacterium diphtheriae that regulates the expression of the diphtheria toxin gene tox, an important virulence factor, as well as other genes important in iron acquisition and homeostasis. DtxR is the prototype for a growing family of iron-regulated bacterial proteins in many pathogenic prokaryotes such as Staphylococcus aureus (1, 11), Treponema pallidum (10, 22), Streptococcus gordonii (13, 15), and Bacillus subtilis (23). In mycobacteria, the iron-regulated transcriptional repressor IdeR (iron-dependent repressor) is a homologue of DtxR (5) and is essential in Mycobacterium tuberculosis (26). IdeR and DtxR show remarkable amino acid similarity (88%) in the first 140 amino acids and are identical in the metal ion binding, DNA binding, and protein-protein interaction domains (34) (Fig. 1). Both proteins contain a helix-turn-helix domain critical for binding to a palindromic DNA sequence that is well conserved (21). Evidence for cross-genus functional complementation has been published previously and shows by gel shift assay that mycobacterial IdeR is able to bind to the corynebacterial tox operator region in a metal cation-dependent manner (27).

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FIG. 1. Amino acid alignment of C. diphtheriae DtxR, M. tuberculosis SirR, and M. tuberculosis IdeR. Heavy line shows the helix-turn-helix domain, with short arrows indicating amino acids important in DNA contact. Triangle-circles show amino acids in the primary metal binding site; long arrows mark those amino acids in the ancillary metal binding site. The bases mutated for this study are outlined in bold.

Using PCR mutagenesis, Sun and colleagues isolated a strain with a point mutation in dtxR that resulted in a single amino acid substitution (glutamic acid to lysine) at position 175 [DtxR(E175K)]. Merodiploid strains containing 1 wild-type copy of dtxR and the mutant allele had a dominant hyperrepressor phenotype under low-iron conditions (29). In a previous paper, we showed by Western blot analysis that this mutant corynebacterial gene could be expressed in M. tuberculosis. When tested in vivo in mice, the DtxR(E175K)-expressing M. tuberculosis strain was attenuated, suggesting that IdeR controls genes essential for virulence in M. tuberculosis (19). The specific IdeR-regulated virulence genes responsible for this attenuation have yet to be fully elucidated; specific genes, such as a siderophore gene (fxbA) (6), a histidine synthesis gene (hisE) (25, 26), Rv3402c, mycobactin genes (mbtA, mbtB, mbtI), and bacterioferritin genes (bfd, bfrA) (9), have been reported to be well regulated.

The amino acid substitution occurs in the C-terminal src homology 3-like (SH3) domain of DtxR (3, 20, 33) that interacts with the polyprolyl tether region (residues 125 to 139) linking the conserved N-terminal with the more divergent C-terminal domain (18, 35). Crystal structures of both DtxR and IdeR have confirmed the structures in the N-terminal domain, including the helix-turn-helix motif that binds the palindromic DNA iron box consensus sequence, ancillary and primary iron binding sites (21), and multiple hydrophobic amino acid residues important for dimer formation. Although the C-terminal domain is less well conserved, crystallography has confirmed that the secondary structures forming the SH3-like fold (7) and its interaction with the ancillary metal ion binding site exist in both DtxR and IdeR (35). Nuclear magnetic resonance analysis has confirmed that the mutant DtxR(E175K) adopts a more ordered conformation through binding of the SH3-like domain to the polypropyl tether region between the N- and C-terminal domains (17).

In this work, we have constructed the IdeR(D177K) mutant, which has a point mutation in the amino acid similar to that in DtxR(E175K). Using a promoterless green fluorescent protein (GFP) construct in Mycobacterium smegmatis and M. tuberculosis, we have shown that the upregulation of IdeR-regulated genes in the absence of iron is abrogated by the presence of IdeR(D177K). IdeR(D177K) also has an iron-insensitive, dominant-positive repressor phenotype. These data are corroborated by reverse transcription-PCR (RT-PCR) showing that both the cognate IdeR mutant and the DtxR mutant result in hyperrepression of IdeR-regulated genes. Taken together, these data suggest that mutant IdeR(D177K) has a mechanism similar to that of DtxR(E175K), stabilizing the intermediate form of the repressor after ancillary metal ion binding and resulting in iron insensitivity through SH3-like domain binding interactions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmids, strains, and culture. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli cultures were grown in LB or Luria agar supplemented with ampicillin (100 µg/ml) or hygromycin (200 µg/ml). M. tuberculosis CDC1551 and M. smegmatis cultures were grown in standard Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI), supplemented with albumin, dextrose, and catalase (Becton Dickinson, Inc., Sparks, MD), 0.1% glycerol, and 0.05% Tween 80 and were incubated at 37°C in roller bottles.

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TABLE 1. Bacterial strains and plasmids used in this study

Construction of IdeR(D177K) integrating vector. The 1.2-kb ideR gene was PCR amplified using Taq polymerase (Sigma-Aldrich, St. Louis, MO) from H37Rv genomic DNA and cloned into a TA cloning vector (Invitrogen, Carlsbad, CA). This plasmid was used as a template to generate the two fragments of ideR on either side of the site to be mutated (codon 177; base pair change from a guanine to an adenosine). The single-base-pair mutation was engineered into the primers for both the left (1-kb fragment using primers ider1 [5'-GGAATTCCTCCGGCATTCCAATCGACAAG] and idermut1 [5'-CGTGATCAGGTCGATTTTGCCCTGAACGTG]) and right (165-bp fragment using primers ider2 [5'-GGAATTCCGCAGGGTAGGTGCGGGTTAGC] and idermut 3 [5'-GAGCACGTTCAGGGCAAAATCGACCTGATC]) sides of ideR. PCR products were gel purified using the QiaexII bead purification system (QIAGEN, Valencia, CA). Equimolar amounts of both fragments were used as a template for a new PCR using ider1 and ider2 as primers. A 1.2-kb product (idermut) was purified and cloned into a TA vector (Invitrogen, Carlsbad, CA). The insert in this plasmid was sequenced to confirm the ideR mutation. A PacI 1.2-kb fragment was purified and ligated into pCK0601 (see below). The integration of the mutated gene into the attB site of M. tuberculosis was confirmed by PCR using primers unique to the pCK0601 vector.

To make pCK0601, plasmid pMH94 (16) was digested with KpnI, filled in with Klenow fragment, and then ligated with PacI linkers (New England Biolabs, Beverly, MA) to make pCK0246. The kanamycin resistance cassette was removed by digesting pCK0246 with HindIII and filling in the ends with Klenow polymerase. A 1.7-kb PstI-BamHI cassette carrying the Streptomyces hygroscopicus hyg gene from p16R1 (8) was cloned into pUC19 to make pHyg1. A 1.9-kb fragment containing the hygromycin cassette was cut from pHJ1 with HindIII and BamHI, filled in with Klenow fragment, and then ligated to pCK0246 with the kanamycin cassette removed as described above to construct pCK0601.

DtxR and IdeR protein overexpression and purification. DtxR protein was overexpressed and purified as previously described (31). IdeR protein was overexpressed in E. coli using the pTrc99 plasmid (Amersham Pharmacia Biotech, Piscataway, NJ) grown in a fermenter with IPTG (isopropyl-ß-D-thiogalactopyranoside) induction in E. coli. Bacteria were harvested by centrifugation. Bacterial pellets were lysed by sonication, centrifuged, applied to a 5.0-cm by 10-cm DEAE-cellulose column, and finally batch eluted with 20 mM Tris-500 mM NaCl (pH 8) buffer (150 ml). The DEAE eluate was loaded onto a Ni²⁺ affinity column (25 ml) and then washed with 20 mM Tris-HCl-500 mM NaCl (pH 8) buffer (100 ml). The bound proteins were then eluted in 20 mM Tris-HCl-2 mM imidazole (pH 8) buffer. The eluate was further purified using a DEAE-Sepharose column (1.6 cm by 20 cm). Purified protein was stored in 20 mM Tris-Cl (pH 7.5)-5 mM dithiothreitol buffer and stored at –80°C until use.

DNA electromobility shift assay. The gel electrophoresis mobility shift assay with purified DtxR (250 ng) and IdeR (250 ng) proteins was performed as previously described (19, 30). Radiolabeled tox promoter-operator region DNA containing the DtxR box was generated by PCR using 100 ng of ³²P-end-labeled primer mixed with 150 ng of unlabeled primer and template DNA from gel-purified 100-bp cold fragments containing the DtxR box PCR amplified with the primers listed in Table 2. Binding reactions were carried out in 10 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 50 mM KCl, 1 mM dithiothreiotol, 5% glycerol, 50 µg/ml calf thymus DNA, and 5 µg of bovine serum albumin. Freshly prepared MnSO₄ was added at 125 µM. For the divalent metal ion-free sample, the divalent metal iron chelator EDTA at a concentration of 0.1 mM was added to the reaction solution. Binding reactions were equilibrated for 30 min. Samples were immediately submitted to electrophoresis at 200 V on a 5% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA buffer.

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TABLE 2. Primers for PCR amplification of iron-dependent promoters

Quantification of iron-dependent promoter activity using a FACS. The original in silico searches to identify candidate genes transcriptionally regulated by IdeR were done with the unannotated sequence of H37Rv by using the DtxR consensus sequence (TTAGGTTAGCCTAACCTTT) and allowing for as many as 4 mismatches. Iron boxes that were within 500 bp of a start codon predicted by MacVector were accepted. Later, when TubercuList (Pasteur Institute database of the M. tuberculosis genome, available at http://genolist.pasteur.fr/TubercuList/) was published, we confirmed those iron boxes that were within 150 bp of a predicted gene and eliminated all others. By using the search tool in TubercuList, all of the iron boxes we tested could be selected by searching for AGG separated by 8 to 9 bp followed by CCT. Promoters containing iron boxes of candidate genes were cloned in front of a promoterless gfpmut3 (enhanced GFP [eGFP]) shuttle vector optimized for fluorescence-activated cell sorting (FACS), pFPV27 (24). Primers are listed in Table 2. Each candidate promoter-GFP vector was transformed into four different bacterial strains: M. smegmatis, M. tuberculosis, M. tuberculosis DtxR(E175K), and M. tuberculosis IdeR(D177K). M. tuberculosis DtxR(E175K) is an M. tuberculosis strain containing an integrated single copy encoding the corynebacterial DtxR(E175K), which has a single amino acid change from glutamic acid to lysine, conferring a positive-dominant phenotype on the parent strain, CDC1551 (19). M. tuberculosis IdeR(D177K) is an M. tuberculosis strain with an integrated copy of the mycobacterial ideR gene mutated in the homologous amino acid, changing the aspartic acid to a lysine. An hsp60 promoter-driven eGFP in pFPV27 was used as a positive control (a kind gift of Lalita Ramakrishnan). Strains were cultivated in glycerol alanine salts medium with or without iron (4). The degree of fluorescence of each bacterial strain in media with and without iron was quantified using FACS (see Fig. 2). Each fluorescence reading was the average of triplicate readings, and each strain was biologically replicated twice. Readings were taken on the day of peak fluorescence of the wild-type strain, which was day 4 for mbtI, Rv1519, and Rv3402c, and day 2 for Rv2123. Ratios were calculated by dividing the peak fluorescence by the baseline fluorescence before iron was depleted. P values could not be determined, because only two biologic replicates were done.

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FIG.2. Flow cytometry of the eGFP plasmid transcriptionally linked to the promoters of mbtI, Rv1519, Rv2123, and Rv3402c and transformed into wild-type M. tuberculosis CDC1551 (black bars), M. tuberculosis DtxR(E175K) (white bars), or M. tuberculosis IdeR(D177K) (grey bars). Each bar represents a ratio calculated as the fluorescence of cells grown in glycerol alanine salts minimal medium in <2 µM iron divided by the fluorescence of cells grown in minimal medium supplemented with 50 µM iron, as measured by flow cytometry. Each bar is the average of two biologic replicates, with each replicate analyzed on the flow cytometer in triplicate.

RT-PCR verification of abrogation of IdeR-regulated gene transcripts with expression of IdeR(D177K). Wild-type CDC1551, M. tuberculosis DtxR(E175K), and M. tuberculosis IdeR(D177K) were grown in Middlebrook 7H9 broth to mid-log phase (optical density at 600 nm [OD₆₀₀], 0.7). Cells were pelleted, washed with phosphate-buffered saline, and then resuspended to an OD₆₀₀ of 0.2 in minimal medium (26) with either low iron (2 µM) or high iron (200 µM). Thirty minutes after growth in iron-poor and iron-rich media, 50 ml of cells was pelleted and suspended in Trizol. RNA was extracted according to previously published methods (14). RNA was reverse transcribed into cDNA. The transcripts were quantified using RT-PCR with the iCycler (Bio-Rad, Hercules, CA) and SYBR green fluorescence. Quantification of the transcripts for the genes of interest Rv1519, mbtI, Rv2123, and Rv3402c was performed. The number of copies of the gene by RT-PCR was calculated based on the standard curve and divided by the number of copies of 16S rRNA. This calculation was done for strains grown under iron-poor conditions and under iron-rich conditions, and the iron-poor values were divided by the iron-rich values to calculate the induction ratio.

Top Abstract Introduction Materials and Methods Results Discussion References

 
GFP reporter assay. Twelve potential IdeR-regulated promoters were identified using MacVector and the TubercuList sequence-searching programs. Because this search was done prior to the publication of other known IdeR-regulated promoters such as fxbA, mbtA, mbtB, bfd, and bfrA, these promoters were not identified by our screen and therefore are not included in our analysis. Using the promoterless eGFP construct, we tested all of these promoters for upregulation in iron-poor minimal medium compared to iron-replete minimal medium. Four promoters were found to be iron regulated in both M. smegmatis and M. tuberculosis by use of the FACS assay. These genes (mbtI, Rv2123, Rv3402c, and Rv1519) are listed with their respective iron boxes in Table 3; Table 4 shows an alignment to the consensus sequence. Four other promoters (hisE, Rv2869, fadE30, Rv0127) constitutively expressed GFP at high levels in M. smegmatis but had iron-regulated expression of GFP in M. tuberculosis by use of our FACS assay. The other three promoters (Rv1396c, Rv2366c, and Rv2417c) were only minimally induced under iron-poor conditions. pheA was minimally induced in both M. smegmatis and M. tuberculosis, but cultures suffered from poor growth and could not be interpreted (Tables 3 and 4).

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TABLE 3. IdeR regulation of selected iron boxes

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TABLE 4. Alignment of the iron boxes in the promoter-operator regions of selected genes

Next we transformed M. tuberculosis DtxR(E175K) and M. tuberculosis IdeR(D177K) with each of the four promoter-GFP constructs that were iron regulated in both M. smegmatis and M. tuberculosis. The relative upregulation of promoter activity was blunted in M. tuberculosis strains expressing either dominant repressor (Fig. 2).

Gel retardation assay with IdeR and DtxR. The same four promoters (mbtI, Rv1519, Rv2123, Rv3402c) also showed divalent cation-dependent binding to both DtxR and IdeR in an in vitro gel retardation assay. By using 250 ng of purified protein, each promoter-operator iron box was retarded by both IdeR and DtxR (Fig. 3). The corynebacterial tox iron box is shown in lanes 1 and 2 as a positive control.

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FIG. 3. Electromobility shift assays. 32P-labeled iron boxes in the promoter-operator regions of the corynebacterial tox gene (lanes 1 and 2), trpE2 gene (lanes 3 and 4), Rv1519 (lanes 5 and 6), Rv2123 (lanes 7 and 8), and Rv3402c (lanes 9 and 10) were incubated in the presence (+) or absence (–) of 250 ng of either IdeR (A and B) or DtxR (C and D). A divalent metal ion (manganese) was added in some reactions (A and C) and omitted from others (B and D).

Non-IdeR-regulated promoters that are iron sensitive. The other three promoters (Rv2869, fadE30, Rv0127) constitutively-expressing GFP at high levels in M. smegmatis, but with iron-regulated expression of GFP in M. tuberculosis by our FACS assay, did not exhibit the same affinity of binding in the gel retardation assay as the first four promoters and bound neither IdeR nor DtxR under high-salt conditions with 250 ng of purified protein. Increasing the protein concentration 10-fold led to binding of both the fadE30 and Rv0127 promoters, however (data not shown). Interestingly, the nucleotide sequence identity between this second set of promoters was also lower. hisE was excluded from this analysis because its promoter-operator region is the same as that of Rv2123.

Transcriptional expression of IdeR-regulated promoters in wild-type M. tuberculosis, M. tuberculosis DtxR(E175K), and M. tuberculosis IdeR(D177K) by RT-PCR. To confirm that the four promoters (mbtI, Rv1519, Rv2123, and Rv3402c) were part of the IdeR regulon and were hyperrepressed in the presence of mutant DtxR(E175K) or IdeR(D177K), we quantified the transcriptional message in both high- and low-iron growth media by using RT-PCR. The expression of the Rv1519, mbtI, and Rv3402c genes relative to the expression of 16S RNA was upregulated under iron-poor conditions. This upregulation was abrogated in the presence of either the mutant IdeR or the mutant DtxR strain constructs, corroborating the FACS data. Consistent data for Rv2123 could not be obtained and are therefore not shown (Fig. 4).

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FIG. 4. Real-time PCR induction of selected genes in M. tuberculosis CDC1551, M. tuberculosis IdeR(D177K), and M. tuberculosis DtxR(E175K). RT-PCR induction ratios, calculated as low-iron message divided by high-iron message normalized for 16S RNA, are shown in black (Rv1519), white (mbtI), and grey (Rv3402c). Error bars, standard deviations for three separate cultures. The Mann-Whitney U (StatView) test was used to compare mutant strains to the wild-type strain. P values of <0.05 are indicated with an asterisk.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent high-resolution crystal data confirm that the SH3-like domain seen in DtxR also exists in IdeR and that the critical amino acid residues in this C-terminal domain are conserved in both structures (35). A multistep model for the activation of apo-DtxR has been suggested by Love et al.; metal ion binding in the ancillary metal ion site leads to stabilization of this conformation with the C-terminal SH3-like domain (18). In the case of the DtxR(E175K) and IdeR(D177K) mutants, electrostatic interactions with the N-terminal domain are postulated to result in a partially ordered structure. Under low-iron conditions, the mutation allows for maintenance of the ordered conformation and prevents the transcription of IdeR-regulated genes. Interestingly, our RT-PCR data support the notion that the presence of low-levels of iron is required for the hyperrepressor phenotype of both DtxR(E175K) and IdeR(D177K) (18). We were unable to see abrogation of the transcriptional expression of the IdeR-regulated genes in the absolute absence of iron (data not shown).

The transcriptional expression of those genes that were iron regulated in M. tuberculosis and constitutively expressed in M. smegmatis was not modulated by the presence of either DtxR(E175K) or IdeR(D177K). Although bearing some homology to the DNA binding consensus sequence of IdeR, these genes did not contain many of the crucial base pairs identified by crystallography (7, 21, 35). One can speculate that the relative affinity of IdeR for various promoter-operator sequences could be a regulatory mechanism. Alternatively, SirR may be involved in the transcriptional regulation of some of these genes. SirR is another mycobacterial DtxR homologue that was first described in staphylococci (11). SirR bears less amino acid similarity to DtxR than IdeR (54%) but shows conserved amino acids in the important regions of the protein, including the metal ion binding sites and the DNA-protein interaction domain (Fig. 1). The critical amino acid residues of the SH3-like domain differ, however. Although SirR appears to control virulence genes in staphylococci, its role in pathogenic mycobacteria remains unclear.

We have shown previously that the dominant-positive DtxR(E175K) mutant was markedly attenuated, especially in later lung infection after the formation of cellular aggregates in mice (19). In addition, IdeR has been shown to be upregulated under acidic conditions in vitro, a finding confirmed in macrophages (12). Iron scavenging by siderophores (mycobactins) is regulated by IdeR, and we demonstrated that the mutant hyperrepressor impairs the ability of M. tuberculosis to upregulate mycobactin production. In this work, we have constructed an M. tuberculosis strain with an integrated copy of the mutant ideR that constitutively represses IdeR-regulated genes. Both the DtxR(E175K) and IdeR(D177K) mutants abrogate the upregulation of the previously described IdeR-regulated genes mbtI, Rv2123, and Rv3402c. We have also confirmed another putative IdeR-regulated gene, Rv1519. Taken together with recent IdeR crystallography data (35) and functional study of the DtxR(E175K) mutant (17), our data corroborate that IdeR(D177K) likely stabilizes the same SH3 domain interaction that results in the hyperrepressor phenotype. There is a growing body of evidence to support IdeR's role in both the in vivo and in vitro survival of M. tuberculosis. This mutant strain will prove useful in further analysis of the IdeR regulon and in the identification of iron-regulated virulence genes. As an essential gene in M. tuberculosis (26), IdeR is a logical drug target to exploit for M. tuberculosis chemotherapy. Drugs that have interfered with mycobactin production in the past, such as PAS (para-aminosalicylate), have been shown to be efficacious.

 
We thank John Love, Robert Harrison, and William Bishai for invaluable advice and help.

This work was supported by funding from the National Institutes of Health (1 K08 AI 01689, 1R01 HL71554) and a grant from the American Lung Association.

 
* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, 1503 E. Jefferson Street, Rm. 108, Baltimore, MD 21231-1004. Phone: (410) 614-6600. Fax: (410) 614-8173. E-mail: ymanabe{at}jhmi.edu.

Editor: J. L. Flynn

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, September 2005, p. 5988-5994, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5988-5994.2005
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