The Mycobacterium Tuberculosis IdeR Is A Dual Functional Regulator That Controls Transcription of Genes Involved in Iron Acquisition, Iron Storage and Survival in Macrophages
Gold B, Rodriguez GM, Marras SA, Pentecost M, Smith I. The Mycobacterium
tuberculosis IdeR is a dual functional regulator that controls transcription of
genes involved in iron acquisition, iron storage and survival in macrophages. Mol Microbiol. 2001 Nov;42(3):851-65. PubMed PMID: 11722747.
Original Title
The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages
Gold B, Rodriguez GM, Marras SA, Pentecost M, Smith I. The Mycobacterium
tuberculosis IdeR is a dual functional regulator that controls transcription of
genes involved in iron acquisition, iron storage and survival in macrophages. Mol Microbiol. 2001 Nov;42(3):851-65. PubMed PMID: 11722747.
The Mycobacterium Tuberculosis IdeR Is A Dual Functional Regulator That Controls Transcription of Genes Involved in Iron Acquisition, Iron Storage and Survival in Macrophages
Gold B, Rodriguez GM, Marras SA, Pentecost M, Smith I. The Mycobacterium
tuberculosis IdeR is a dual functional regulator that controls transcription of
genes involved in iron acquisition, iron storage and survival in macrophages. Mol Microbiol. 2001 Nov;42(3):851-65. PubMed PMID: 11722747.
functional regulator that controls transcription of genes
involved in iron acquisition, iron storage and survival in macrophages Benjamin Gold, 1,2 G. Marcela Rodriguez, 2 Salvatore A. E. Marras, 2 Mickey Pentecost 3 and Issar Smith 1,2 * 1 Department of Microbiology, New York University Medical Center, New York, NY 10016, USA. 2 TB Center, Public Health Research Institute, 455 First Avenue, New York, NY 10016, USA. 3 Albert Nerkin School of Engineering, Cooper Union, New York, NY, USA. Summary In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-depen- dent regulator), an iron-responsive DNA-binding pro- tein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI ), aromatic amino acids ( pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identied in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like mol- ecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtAmbtB, mbI, rv3402c and bfrAbfd, for interaction with IdeR and for iron- dependent expression. Gel retardation experiments and DNase footprinting analyses with puried IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the 210 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse tran- scriptasepolymerase chain reaction (RTPCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtI and rv3402c, were induced during M. tubercu- losis infection of human THP-1 macrophages. Introduction The low levels of available iron in a bacterial pathogens mammalian host (estimated at 10 218 M; Neilands, 1995) may potentiate the expression of genes encoding iron acquisition systems, virulence factors and toxins (Muller et al., 1983). Iron limitation by the human body is exacerbated by an upregulation of the expression of transferrin, lactoferrin, ferritin and other iron-sequestering molecules in response to bacterial infections (Leffel and Spitznagel, 1975; Wright and Gallin, 1979; Muller et al., 1983; Weinberg, 1984). These molecules have a high afnity for iron and sequester available free iron required for bacterial growth. Therefore, many bacterial pathogens, such as Yersinia, Salmonella and Legionella, require iron acquisition systems to survive the iron-limited environment of their host (Pope et al., 1996; Bearden and Perry, 1999; Janakiraman and Slauch, 2000). Recent results suggest an essential role for iron acquisition in Mycobacterium tuberculosis, the aetiological agent of tuberculosis, during infection. A null mutation of mbtB in M. tuberculosis, encoding an enzyme involved in the biosynthesis of the siderophore mycobactin (Quadri et al., 1998), was shown to have impaired growth in the human macrophage cell line THP-1 (De Voss et al., 2000). Furthermore, mbtB expression is induced up to 60-fold during M. tuberculosis infection of mice (J. Timm et al., unpublished). These data imply a crucial role for mycobactin-mediated iron acquisition for M. tuberculosis virulence in both macro- phage and murine models. If M. tuberculosis faces low-iron conditions in a host, we can hypothesize that Accepted 29 August, 2001. *For correspondence at the TB Center. E-mail smitty@phri.nyu.edu; Tel. (11) 212 578 0868; Fax (11) 212 578 0804. Molecular Microbiology (2001) 42(3), 851865 Q 2001 Blackwell Science Ltd many iron-regulated genes will be upregulated in the course of an infection, and that these genes could be important for the tubercle bacillus adaptation and survival in the host. Bacterial iron acquisition has been shown to be regulated by the iron-responsive proteins of the Fur and DtxRfamilies. IdeR (iron-dependent repressor), found in all mycobacteria (Doukhan et al., 1995), is the structural and functional homologue of the Corynebacterium diphtheriae DtxR (Schmitt et al., 1995) and has been shown in Mycobacterium smegmatis to negatively regulate siderophore biosynthesis (Dussurget et al., 1996). A functional IdeR binding site (an IdeR box) was found upstream of fxbA, and three other putative IdeR binding sites were identied in the M. smegmatis exochelin biosynthesis locus (Fiss et al., 1994; Yu et al., 1998; Dussurget et al., 1999). The M. smegmatis ideR mutant cannot mount an effective oxidative stress response (Dussurget et al., 1996), probably because of decreased expression of katG and sodA as the levels of both mRNA and protein decline (Dussurget et al., 1996; 1998). These results suggest that IdeR is a functional homologue of Fur, a pleiotropic regulator controlling over 60 genes in Escherichia coli involved in diverse functions Table 1. Putative IdeR binding sites located in the M. tuberculosis H37Rv genome. The putative IdeR box sequences in this table were identied by probing the M. tuberculosis genome with a consensus IdeR/DtxR binding sequence using the program TUBERCULIST. Genes that have been grouped together within a black box indicate those that are divergently transcribed from the same IdeR box. The IdeR binding sites of fxbA, from M. smegmatis, and hisEirg2, from M. tuberculosis, are the only IdeR targets that have been biochemically and physiologically veried to date (Dussurget et al., 1999; Rodriguez et al., 1999). a. Mis., mismatches from the consensus sequence. b. Rv#: indicates the numerical designation of each open reading frame in the annotated genome. The c that follows some Rv#s indicates that the gene has a reverse orientation. An asterisk (*) indicates IdeR boxes that have been validated. c. Bases with a black background match the query sequence. The letter W represents weak basepairing (the bases A or T), and S is for strong basepairing (G or C). d. Location indicates the distance of the 5 0 end of the IdeR box to the predicted ORF start site. 852 B. Gold et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 ranging across iron homeostasis, oxidative stress, toxin synthesis and general metabolism (Hantke, 2001). However, the entire IdeR regulon has yet to be dened, nor have any direct positive targets been identied. In this work, we have identied IdeR-regulated genes in M. tuberculosis using a computer-based approach to search the recently published genome sequence (Cole et al., 1998). Four promoter regions containing putative IdeR boxes were shown to bind puried IdeR, and DNase protection analysis showed that IdeR protects the predicted IdeR box sequence. The transcription of the six genes downstream of these IdeR boxes (mbtA, mbtB, mbtI, rv3402c, bfd and bfrA) was induced by growth in an iron-limited medium. In addition to IdeRs role as a repressor, we provide evidence that IdeR activates bfrA transcription directly. In addition, three IdeR-repressed genes were found to be induced during growth of M. tuberculosis in human THP-1 macrophages. Results Identication of IdeR boxes IdeR was predicted to bind a similar DNA sequence to the C. diphtheriae DtxR, given the following observations: the DNA-binding domain of IdeR is 92% identical and 100% similar on the amino acid level with that of DtxR (Doukhan et al., 1995); these proteins have almost identical crystal structures (Pohl et al., 1999); IdeRcan complement a dtxR mutant for iron-dependent regulation of DtxR-regulated promoters (Schmitt et al., 1995); and nally, IdeR binds to the C. diphtheriae tox promoter and other DtxRtarget gene promoters (Schmitt et al., 1995). Therefore, we hypoth- esized that many of the IdeR boxes in the M. tuberculosis genome could be identied by conducting a computer search using a DtxR binding site sequence as a surrogate. A preliminary search for IdeR boxes using the BLAST program against every published sequence (not specic for M. tuberculosis ) resulted in the identication of the M. tuberculosis hisEirg2 promoter region (Rodriguez et al., Fig. 1. IdeR binds DNA fragments containing the predicted IdeR box. IdeR was added in fourfold increasing concentrations, from 0.06 to 1 mM, to 32 P-labelled DNA probes in the presence of 200 mM Ni 21 , and complexes were resolved on an 8% Tris acetate polyacrylamide gel containing 200 mM Ni 21 . The probe alone without added protein is in the left-most lane and indicated by (). Promoter regions containing one iron box included mbtAmbtB (A), mbtI (B) and rv3402c (C), whereas bfrAbfd (D) contains two iron boxes. Control gel retardation experiments using DNA fragments without an IdeR box showed no interaction with the IdeR protein (data not shown). U, unbound probe in the absence of IdeR; B1, B2 and B3, bound probe in the presence of IdeR. Identication of IdeR-regulated genes in M. tuberculosis 853 Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 1999). We have nowconducted a broader search of the M. tuberculosis published genome (Cole et al., 1998) for IdeR boxes to identify genes regulated by IdeR using the available computer program TUBERCULIST (http://genolist. pasteur.fr/TubercuList/ Moszer et al., 1995) (Table1). The criteria chosen as qualities for a DNA region regulated by IdeR were limited to those within 1250 to 1100bp of the predicted start codon of an open reading frame (ORF) and containing ve or fewer mismatches from our query sequence of TWAGGTWAGSCTWACCTWA (where WA or T, and SG or C). There are two major reasons why the putative IdeR boxes in Table 1 are limited to ve mismatches. First, there was a dramatic increase in potential IdeR binding sites with each subsequent mismatch allowed (estimated as more than 100 targets for six mismatches and more than 700 targets for seven mismatches). Secondly, if IdeR boxes were randomly distributed in the M. tuberculosis chromosome, there should be approximately four times more putative IdeR boxes inside an average length ORF (<1000 bp) compared with a 250bp promoter region. This was not the case, as there was only a small number of putative IdeR binding sites with four mismatches (two in ORFs versus 17 located 250 bp upstream of the putative translational start site), whereas ve mismatches resulted in 16 in ORFs versus 19 located 250bp upstream of the putative translational start site. Although this indicated that the computer search for IdeR boxes was probably more accurate with #four mismatches, putative binding sites with ve mismatches were left in Table 1, as some of these were located upstream of genes whose transcription was predicted to be regulated by IdeR (annotated to be involved in iron storage and acquisition). Over 40 genes preceded by putative IdeR boxes were Fig. 2. IdeR binds to predicted IdeR box sequences. 32 P-labelled fragments were bound to 1 mM puried IdeR in the presence of 200 mM Ni 21 and subjected to DNase digestion. MaxamGilbert A1G sequencing reactions were performed on the same probes to identify the exact sequence to which IdeR binds. Reaction products were separated on a 6% TBE polyacrylamide sequencing gel containing 8 M urea. Protected regions are marked with a black line, and a summary of these sequences is shown in Fig. 3. A. mbtA top strand. B. mbtB top strand. C. mbtI bottom strand. D. mbtI top strand. E. rv3402c bottom strand. F. rv3402c top strand. G. bfd top strand. H. bfrA top strand. A/G, MaxamGilbert A1G sequence ladder; , no IdeR added, 1, 1 mM IdeR. 854 B. Gold et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 identied and fell into several categories (Table 1). One group encodes proteins that are annotated to be directly involved in either iron acquisition (mbtAmbtB, mbtI, rv1348, rv1347c ) or iron storage (bfrA, bfrB). The second group of genes, encoding annotated proteins involved in amino acid biosynthesis (hisB, hisB-like, pheA, lysyl tRNA) may be involved in siderophore biosynthesis. The last group contains many genes that either have no obvious relationship to iron (murB, acp) or have no signicant homologies to any known proteins in databases. To test the functionality of the IdeR boxes identied by the computer search, we analysed four that were between or in front of the following genes: mbtAB, mbtI, rv3402c and bfdbfrA. mbtA, mbtB and mbtI encode enzymes involved in the biosynthesis of mycobactin, the major M. tuberculosis siderophore (Quadri et al., 1998; De Voss et al., 2000). rv3402c encodes a protein with .40% amino acid similarity to three types of enzymes: an aminotrans- ferase, a dehydratase and an enzyme involved in perosamine/O-antigen biosynthesis. bfrA encodes a bacterioferritin subunit that is involved in iron storage. bfd is not currently annotated in the M. tuberculosis genome, but we found that this ORF has homology (30 40% amino acid identity, .50% conserved amino acids) to Bfd from Escherichia coli, a protein that associates with BfrA and may have a role in donating iron to the bacterioferritin complex (Garg et al., 1996; Quail et al., 1996). In M. tuberculosis, bfd and bfrA are adjacent and divergently transcribed (Fig. 5C) and, in E. coli, bfd and bfrA are also neighbouring genes but are transcribed in the same direction (Andrews et al., 1993). IdeR binds to DNA fragments containing putative IdeR boxes To demonstrate that IdeR could bind to the putative IdeR boxes identied from our computer search, DNA frag- ments containing these sequences were PCR amplied and labelled with 32 P for gel retardation analysis. The labelled probes were mixed with various concentrations of puried IdeR (0.061 mM) and then separated on 8% polyacrylamide gels (Fig. 1). Nickel was used as the divalent metal in the binding reactions on account of its redox stability compared with ferrous iron. The specicity of puried IdeR to its target sequences has been demonstrated in a previous work (Rodriguez et al., 1999). In addition, we have shown previously IdeRs broad in vitro divalent metal specicity and IdeRs metal dependence for function (Schmitt et al., 1995; Dussurget et al., 1996). The migration of all four DNA fragments tested was retarded by IdeR in a concentration-dependent manner (Fig. 1). The labelled probes from mbtAB, mbtI and rv3402c, with one IdeR box, had one major shifted product (B2), whereas bfdbfrA, containing two IdeRbinding sites, had two major shifted products (B2 and B3). All four fragments exhibited one minor intermediate band (B1) that appeared with the lower concentrations of IdeR, which may represent an intermediate with one IdeR dimer bound to the probe (White et al., 1998). These results indicate that IdeR can bind to these DNA fragments that contain putative IdeR-binding sequences. IdeR binds to IdeR box sequences We performed DNase protection assays with IdeR bound to the four DNA fragments containing putative IdeR boxes, mbtAB, mbtI, rv3402c and bfdbfrA, in order to identify the DNA sequences bound by the protein. The results of the DNase footprinting experiments are shown in Fig. 2, and the sequences protected by IdeR are summarized in Fig. 3. IdeR protected the predicted IdeR box sequence for mbtAmbtB, mbtI, rv3402c and both IdeR boxes for bfd bfrA. The protection pattern covers the 19 bp IdeR box sequence and also includes up to a 10 bp overhanging region of protection 3 0 to the end of the IdeR box on both the top and the bottom strand. The sequences protected by IdeR are similar to those found in our previous studies with the M. smegmatis fxbA and the M. tuberculosis hisE irg2 (Dussurget et al., 1999; Rodriguez et al., 1999), and these are included here for comparison (Fig. 3). IdeR functions as a repressor by blocking the 210 region of the promoter We mapped the transcriptional start points (TSP) of mbtB, mbtI, rv3402c, bfd and bfrA by primer extension analysis using RNA from M. tuberculosis grown in either low- or high-iron-containing medium (Figs 5 and 6; data not shown), and the results are summarized in Fig. 3. The mRNA levels of mbtA were too low for this analysis. The TSP of mbtB, mbtI, rv3402c, bfrA and bfd all mapped within the IdeR box or 5 bp downstream of the box. We identied putative 210 sequences with similarity to the sequence TAgRcT (Gomez and Smith, 2000) that were the correct distance from the TSPs (Fig. 3). The mapping of the TSPs indicates that these IdeR boxes are located in close proximity to the 210 region, which ts a model that, in the presence of iron, IdeR functions as a repressor by occluding the site of RNApolymerase binding (Fig. 8), as is the case for DtxRbinding to its target genes (Schmitt et al., 1992). Genes preceded by an IdeR box are regulated by iron The expression of genes with IdeR boxes was analysed in M. tuberculosis cultures grown with low and high levels of Identication of IdeR-regulated genes in M. tuberculosis 855 Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 iron by reverse transcriptasepolymerase chain reaction (RTPCR) coupled with molecular beacons (mbRT PCR) (Manganelli et al., 1999). We used the sigA mRNA for normalization, as its levels remain constant in low- and high-iron concentrations (data not shown; Dussurget et al., 1999; Rodriguez et al., 1999). The average ratio of mRNA copies in low/high iron were 14 (mbtA), 49 (mbtB), 17 (mtbI ), 17 (rv3402c ) and 40 (bfd) (Fig. 4). The effect of iron on bfrA expression is described in the next section. As was predicted from the presence of an IdeR box in their promoter region, the transcription of these genes was indeed repressed by high iron levels. IdeR acts positively on bfrA expression We expected that the expression of bfrA, encoding an iron storage protein, bacterioferritin, would be upregulated in high-iron conditions as has been shown in other bacteria (Andrews, 1998; Miller et al., 2000). However, as all the Fig. 3. Summary of data from DNase footprinting and transcriptional start point analysis. The predicted IdeR binding sequence is indicated in capital letters, and the region on either strand protected fromDNase digestion by IdeRis indicated by shading with black. Transcriptional start points (TSPs) for each gene were mapped by primer extension and veried with two separate primers, using RNA prepared from M. tuberculosis grown in low- and high-iron medium. Data for the bfrA TSPs are shown in Fig. 5; others are not shown. The TSP has not yet been mapped for mbtA. For comparison, data on IdeRs interaction with the promoters of the M. smegmatis fxbA and the M. tuberculosis hisEirg2 and their TSPs in low iron have been included (Dussurget et al., 1999; Rodriguez et al., 1999). Fig. 4. Expression of genes preceded by IdeR boxes during iron limitation. RNA was prepared fromM. tuberculosis H37Rv after growth in a minimal medium containing 50mM FeCl 3 or lacking iron for six generations. Using gene-specic molecular beacons, low-/high-iron induction ratios were calculated with quantitative molecular beacon RTPCR. Gene expression was normalized to sigA, whose mRNA levels are not affected by iron concentration. 856 B. Gold et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 other IdeR- and DtxR-regulated genes have been shown to berepressedbyhighironlevels(Schmitt et al., 1992; Schmitt and Holmes, 1994; Lee et al., 1997; Dussurget et al., 1999; Rodriguez et al., 1999), it was intriguing to study how bfrA would be regulated by IdeR. In addition, the bfrApromoter is unlike the other IdeR-regulated genes because it has two tandemIdeR boxes, beginning at 207 and 231bp upstream of the annotated ORF start site (Fig. 5). Fig. 5. bfrA low- and high-iron promoters. RNA from a culture grown in conditions of iron limitation was isolated from both M. tuberculosis and M. smegmatis carrying an M. tuberculosis bfrAlacZ fusion. The 5 0 mRNA termini were mapped using 32 P-labelled reverse primers specic to the M. tuberculosis bfrA mRNA. The TSPs were identical when using total RNA isolated from M. tuberculosis H37Rv or M. smegmatis MC 2 155 harbouring the M. tuberculosis bfrAlacZ: (A) P high using RNA from cells grown in a medium containing high iron; and (B) P low1 and P low2 , using RNA from cells grown in an iron-limiting medium. The locations of P low1 , P low2 and P high in the bfrA upstream region are shown in (C), where the two IdeR boxes are identied by bold lettering. Identication of IdeR-regulated genes in M. tuberculosis 857 Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 As we speculated that bfrA might be positively regulated, we wanted to determine whether the expression of this gene required IdeR and the two IdeR binding sites. To address these questions, a bfrAlacZ fusion construct was made that contained 378bp upstream from the bfrA initiating codon and was integrated into the chromosome of wild-type M. smegmatis MC 2 155 and its isogenic ideR mutant, SM3 (Dussurget et al., 1996). In addition, a bfrAlacZ fusion was made with the tandem IdeR boxes deleted (bfrADIBlacZ), and this construct was also incorporated into MC 2 155 and SM3. Initial experiments using the intact bfrAlacZ fusion integrated into M. smegmatis MC 2 155 demonstrated that levels of bfrA decreased (<twofold) during iron limitation. When the bfrAlacZ and bfrADIBlacZ constructs were integrated into M. smegmatis SM3, there was little or no Fig. 6. IdeR and iron-dependent regulation of bfrA. A map of the bfrA promoter region is summarized in (A), showing the location of the two IdeR boxes (IB) and P low1 , P low2 and P high . The expression of P high in high and low iron was analysed using M. smegmatis strains carrying bfrAlacZ (B) and a strain carrying a bfr lacZ with two IdeR boxes deleted, bfrADIBlacZ (C). P low expression in high and low iron was analysed only using the bfrAlacZ (F), as the deletion of the IdeR boxes abrogates P low activity. The regulation of bfrA was analysed by mbRTPCR using a beacon that recognizes a sequence common to the mRNAs produced by P low and P high , and recognizing a sequence 5 0 to P high and unique to P low (E). 858 B. Gold et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 lacZ expression (data not shown). The analysis of the bfrA promoter region was extended to locate the TSP, using RNA isolated fromthe M. smegmatis strains containing the M. tuberculosis bfrAlacZ and bfrADIBlacZ fusions. The primers used were specic for the M. tuberculosis bfrA and not endogenous M. smegmatis bfrA, as the primer extension products were not seen using RNA isolated from M. smegmatis not containing the M. tuberculosis promoter fusions (data not shown). The TSPs of bfrA, from both M. tuberculosis total RNA and the M. smegmatis strain carrying the M. tuberculosis bfrAlacZ fusion were identical when grown in both high iron (Fig. 5A) and low iron (Fig. 5B), and the location of these TSPs is shown in Fig. 5C. The stronger band intensity of the M. tuberculosis P low1 and P low2 in Fig. 5B results from longer iron starvation. These results indicated that the regulation of the M. tuberculosis bfrA could be studied using M. smegmatis as a surrogate host. A TSP was detected in high iron and was located 110bp upstreamof the ATG(P high ) (Figs 5A and 6B). P high activity is decreased during iron starvation (Fig. 6B, lanes 1 and 2) and is dependent on IdeR, as the levels of P high transcript are very low in the ideR mutant strain (Fig. 6B, lanes 3 and 4). If the IdeR boxes are deleted (bfrADIBlacZ), the expression of P high in high iron is reduced (Fig. 6C, lanes 1 and 2) compared with when the IdeR boxes are present (Fig. 6B, lanes 1 and 2). A combination of deleting the IdeR boxes and lacking IdeR completely abrogated bfrA P high activity (Fig. 6C, lanes 3 and 4). The mRNA produced by P high is not a processed mRNA produced by P low , as it is made, albeit at lower levels, even when P low is deleted (Fig. 6C, lanes 1 and 2). Two primer extension products were present only during iron starvation, which mapped to the distal IdeR box (P low1 and P low2 ) (Figs 5B and 6D). P low1 and P low2 were always present in equal amounts and will henceforth be named P low to refer to both promoters. The repression of P low in high iron is dependent on IdeR, as the transcription of P low remains high in the ideR mutant (Fig. 6D). The activity of P high was estimated to be 510 times stronger than P low by quantitative RTPCR and band intensity from primer extension (data not shown). The contributions of P low and P high were measured more precisely using mbRTPCR of M. tuberculosis RNA derived from cultures grown in low- and high-iron medium. The total amount of bfrA mRNA (P low 1P high ) decreased in a time-dependent manner to about 30% during iron starvation (Fig. 6E). Using RTPCR with a beacon specic for the P low mRNA (which recognizes a sequence 5 0 to P high ), P low was demonstrated to be induced about 11-fold during iron starvation (Fig. 6E). The decrease in bfrA mRNA (P low 1P high ) during iron starvation can be attributed to a combination of mRNA differentially expressed from both P low and P high (Fig. 8). P high is expressed during growth in conditions of iron excess and requires IdeR for expression. During conditions of iron starvation, P high is shut off, and IdeR releases its repression of P low , allowing for low-level expression from P low . IdeR-controlled genes are derepressed during M. tuberculosis infection of human macrophage-like THP-1 cells The levels of mbtB, mbtI, Rv3402c and bfrA were compared in broth cultures of M. tuberculosis and M. tuberculosis infection of the human monocytic cell line THP-1 that had been differentiated into macrophages (Fig. 7). THP-1 macrophages were infected with M. tuberculosis and, at various time points, macrophages containing internalized M. tuberculosis were harvested, and RNA was isolated for subsequent analysis by mbRT PCR. sigA mRNA was used to normalize all ratios, as it had been determined previously that the M. tuberculosis sigA mRNA levels remain constant during M. tuberculosis infections of THP-1 cells (Manganelli et al., 2001; E. Dubnau et al., submitted). The expression of genes previously reported to be expressed during intracellular growth in macrophages, icl, encoding isocitrate lyase (McKinney et al., 2000), and hspX, encoding a-crystallin (Yuan et al., 1998), was analysed, and both were found to be strongly induced (data not shown; E. Dubnau et al., submitted). Two genes from the mycobactin biosynthesis operon were each induced to essentially the same levels at both 24 and 72 h: mbtB (<38-fold) and mbtI (<fourfold) (Fig. 7). rv3402c was induced 11-fold by 24 h, and 25-fold Fig. 7. IdeR-regulated genes during M. tuberculosis infection of THP-1 macrophages. THP-1 macrophages were infected at a multiplicity of infection of <1 with M. tuberculosis H37Rv, and total RNA was extracted at 24 and 72 h after infection. mRNA levels were determined by mbRTPCR, and values were normalized against sigA, whose levels remain constant during M. tuberculosis growth in vitro, during iron starvation and in macrophages. Identication of IdeR-regulated genes in M. tuberculosis 859 Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 by 72 h (Fig. 7). The levels of bfrA (P low 1P high ) did not change signicantly from broth-grown cultures at either time point. To control for induction of gene expression during the 2 h period while the macrophages were ingesting M. tuberculosis, the levels of mRNA for all the genes were compared with cultures incubated in RPMI or 7H9 for 2h. Only mbtB was responsive to a 2 h exposure to RPMI and was induced threefold, and the other genes were unaffected. Accordingly, if mbtB is normalized against RNA from M. tuberculosis exposed to RPMI for 2 h, its induction is 11-fold (versus 38 if normalized to 7H9). The induction of the mycobactin biosynthesis genes, mbtB and mbtI, suggests that the mycobacterial phagosome may be limiting for iron, consistent with previous ndings (De Voss et al., 2000). Discussion We identied putative IdeR-regulated genes as the result of a computer search for IdeRbox-like sequences in the M. tuberculosis genome. Six genes selected to perform DNA- binding experiments and quantitative analysis of gene expression were all regulated by iron levels and IdeR. This suggests that many of the other putative IdeR targets may behave in a similar fashion (Table 1), but this conclusion must be validated by further experimentation. Data presented in this communication and in previous results (Dussurget et al., 1996; Rodriguez et al., 1999) show that IdeR is a pleiotropic regulator in M. tuberculosis and M. smegmatis, making it a functional homologue of Fur in non-actinomycete bacteria. IdeR has been shown previously to control siderophore biosynthesis in M. smegmatis (Dussurget et al., 1996), and we present evidence that, in M. tuberculosis, IdeR directly affects the transcription of the mbtA, mbtB and mbtI. It is likely that other genes in the mbt cluster (mbtAH, lipK) are co- transcribed with mbtA, mbtB and mbtI during iron starvation. Mycobacterial siderophores are synthesized by non-ribosomal peptide synthases and contain deriva- tized amino acids (Quadri et al., 1998;De Voss et al., 1999; 2000; Quadri, 2000). As we nd IdeR boxes upstream of genes presumably involved in the biosynthesis of amino acids such as histidine (hisE, hisB-like) and phenylalanine ( pheA), it is possible that these aromatic amino acids are also used in siderophores or compounds with siderophore- like activity. Although IdeRhas been shown to control katG and sodA indirectly in M. smegmatis, we did not nd IdeR boxes upstream of genes annotated as oxidative stress detoxifying enzymes; however, a putative IdeR binding site with six mismatches was identied upstream of sodC (data not shown). The mechanism of transcriptional control of iron storage in bacteria has yet to be completely elucidated. In E. coli, a putative 5 0 Fur box was identied upstream of bfrA (Andrews et al., 1989), and this gene was shown to be positively regulated by iron (Andrews, 1998). However, no direct binding of Fur to this putative Fur box has been observed (Stojiljkovic et al., 1994). bfrA transcription in Pseudomonas aeruginosa increases during growth in high-iron medium and also by exposure to hydrogen peroxide (Ma et al., 1999). ftn, encoding ferritin, another iron storage molecule in E. coli, is also positively regulated by Fur (Abdul-Tehrani et al., 1999), but no direct interaction with Fur has been observed, either. Thus, Fur can activate the transcription of genes involved in iron storage, but it is currently unknown whether this effect is direct or indirect. Intriguingly, bfrA in M. tuberculosis is controlled by three promoters, two are IdeR and iron repressed (P low1 and P low2 ), whereas the other is activated by IdeR and high levels of iron in the growth medium (P high ) (Fig. 5). The total amount of bfrA mRNA decreases approximately threefold during iron starvation, but this level is a sum of the mRNA produced by the P low and P high : during iron starvation, P low is activated and P high is repressed (Figs 5 and 6B, D and E). Eliminating the IdeR boxes upstream of bfrA eliminates P low and signicantly lowers, but does not eliminate, P high transcripts, which indicates that P high is a Fig. 8. Model of IdeRfunction in M. tuberculosis. Our data indicate that IdeR functions as a repressor of transcription by blocking the 210 region of IdeR-regulated promoters, which blocks RNA polymerase (RNAP) from binding and initiating transcription. During iron starvation, IdeR dissociates from its IdeR box sequence, and RNAP initiates transcription of both bfd and the bfrA P low1,2 . In the presence of iron, IdeR blocks transcription of bfd and bfrA P low1,2 and plays an as yet uncharacterized role in the activation of the bfrA P high . 860 B. Gold et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 true promoter, and its mRNA is extremely unlikely to be a cleavage product of the P low transcript (Fig. 6C). Although IdeR and its binding site are necessary for maximal expression of bfrA, the fact that some bfrA expression is observed in the absence of IdeRboxes must be explained. We favour the hypothesis that IdeR, bound to the two IdeR boxes, contacts and activates RNA polymerase directly to transcribe bfrA (Fig. 8). It was shown recently that MntR, another member of the DtxR family, can both positively and negatively regulate the expression of manganese uptake genes in Bacillus subtilis (Que and Helmann, 2000). Thus, like Fur, DtxR-like proteins appear to have positive roles in transcription (Vasil and Ochsner, 1999). In the absence of the IdeR boxes, IdeR may still contact RNA polymerase, but less efciently than when bound to the IdeR boxes. This hypothesis is currently being tested. Our data suggest two potential explanations why bfrA mRNA is produced under both low- and high-iron conditions. The simplest model is that M. tuberculosis produces low levels of BfrA during iron starvation to maintain iron homeostasis during a shift to an iron-replete medium. The second model is that regulation of iron storage in M. tuberculosis is post-transcriptional. This is the case in eukaryotes, in which ferritin mRNA is transcribed in low iron, but translation is blocked by the binding of an IRP (iron- regulatory protein, a cis-aconitase) to the 5 0 IRE of ferritin mRNA (Klausner et al., 1993). In this model, bfrA mRNA is expressed during iron starvation to create an mRNA pool, which can be rapidly translated and allows cells to store excess iron if a shift to high iron occurs. In support of this model, reports have shown that aconitases in prokaryotes can bind mRNA (Tang and Guest, 1999), and M. tuberculosis has a cis-aconitase (rv1475c ). In addition, the sodB mRNA half-life was decreased in a fur mutant, suggesting that Fur has a role in post-transcriptional regulation (Dubrac and Touati, 2000). We are currently investigating the possibility of post-transcriptional regu- lation of bfrA. Preliminary data from our laboratory indicate that the M. tuberculosis ferritin homologue, bfrB (rv3841), is also positively regulated by IdeR, and this mechanism is currently under investigation (data not shown). Many of the potential IdeR-regulated genes identied in this study have no obvious relationship to iron metabolism. The possibility that genes involved in lipid and membrane biosynthesis (acpP, murB) are regulated by iron raises an intriguing possibility that M. tuberculosis alters its membrane structure under iron-limiting conditions, such as those encountered during infections. This type of adaptation has been seen in Salmonella, which was shown to alter its peptidoglycan structure during infection of epthithelial cells (Quintela et al., 1997). The phago- somes of Salmonella and Legionella have been shown to be limiting for iron, estimated at 1 mM (Byrd and Horwitz, 1989; Garcia-del Portillo et al., 1992; Mahan et al., 1995). There is evidence that M. tuberculosis encounters an iron- limited environment during intracellular growth, as myco- bactin is required for growth in macrophages (De Voss et al., 2000). M. tuberculosis faces low-iron conditions during infections in mice, as levels of mbtB mRNA increase <60-fold and bfrA levels decrease 10-fold in lungs (J. Timm et al., submitted). In addition, M. tuberculosis virulence was attenuated in mice when the bacterium contained a mutated dtxR allele that was previously shown to behave as a constitutive repressor of C. diphtheriae genes during iron limitation in an E. coli surrogate system(Sun et al., 1998; Manabe et al., 1999). It was hypothesized that the mutated DtxR was decreasing M. tuberculosis iron acquisition during a mouse infection, although this has not been shown directly (Manabe et al., 1999). In the work reported here, we describe the expression of iron- and IdeR-regulated genes during M. tuberculosis infection of THP-1 macrophages, and these results suggest that M. tuberculosis faces an iron-limiting environment in the macrophage. Therefore, many of the genes identied by the computer search for IdeR boxes are good candidates for inactivation and subsequent study of their roles in virulence. Experimental procedures Identication of IdeR iron boxes An initial low-stringency search of the M. tuberculosis genome using the BLAST program, querying for the DtxR/IdeR binding sequence, identied one promoter region of the divergently transcribed genes, hisEirg2, which was in fact regulated by IdeR (Rodriguez et al., 1999). In this work, various combinations of the consensus DtxR iron box were used to probe the M. tuberculosis H37Rv published sequence (Cole et al., 1998) with the program TUBERCULIST (http://genolist. pasteur.fr/TubercuList/) (Moszer et al., 1995). The query sequence used was TWAGGTWAGSCTWACCTWA (where WA or T and SG or C), and we allowed for up to ve mismatches and limited the search to within 2250 to 1100bp of each ORFs predicted start codon. Bacterial strains, media and growth conditions Escherichia coli strains JM109 and XL1-Blue (Stratagene) were used for cloning and grown in LuriaBertani (LB) broth. Wild-type M. smegmatis mc 2 155 and the M. smegmatis ideR strain SM3 were grown in Middlebrook 7H9 liquid medium supplemented with 0.2% glycerol and 0.05% Tween 80, and M. tuberculosis strain H37Rv was grown in the same medium with the addition of 0.5%bovine serumalbumin (BSA) fraction V, 0.2%dextrose and 0.085%NaCl (ADC supplement). When required, media were supplemented with 20 mg ml 21 strepto- mycin, 10 mgml 21 kanamycin and 100mgml 21 ampicillin. To assay gene expression by mbRTPCR or primer extension analysis in low and high iron, M. tuberculosis was grown in MMT-ADN (minimal medium with 0.05% Tween 80, treated Identication of IdeR-regulated genes in M. tuberculosis 861 Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 overnight with Chelex 100; Bio-Rad) supplemented with either 0 or 50 mM FeCl 3 . Final concentrations of iron in low-iron MMT-ADN have been shown previously by atomic absorption spectroscopy to be in the range of ,1 mM iron (Rodriguez et al., 1999). In order to achieve conditions of iron starvation, M. tuberculosis H37Rv was passaged for two generations in low-iron medium and then diluted to an optical density of 0.05 in the same medium lacking iron or in a high-iron medium. b-Galactosidase assays b-Galactosidase assays were performed as described previously (Miller, 1972; Dussurget et al., 1999). Briey, M. smegmatis was grown in LB mediumsupplemented with 0.1% Tween to mid-late logarithmic phase (OD 600 0.40.6), which was made iron limiting by the addition of 2,2 0 -dipyridyl to 200mM and grown for an additional 3h. bfrAlacZ constructs A 430bp construct encompassing 2378 to 152 of bfrA was amplied by PCR, cloned into the vector psM128 and integrated in single copy into the att site of M. smegmatis wild type (MC 2 155) and ideR mutant (SM3) (Dussurget et al., 1999). A construct was made by deleting the two iron boxes (2255 to 2199), resulting in bfrADIBlacZ, which contains an additional XhoI site. Constructs were veried by sequencing. IdeR purication IdeRwas overexpressed in M. smegmatis, and the protein was puried by techniques similar to those described previously but with some modications (Schmitt et al., 1995; Pohl et al., 1999). The wild-type copy of the M. tuberculosis ideR was cloned downstream of the L5 mycobacteriophage promoter, P left , and then placed into pSM232 (pMV261 1 streptomycin/spectino- mycin resistance cassette), resulting in pSM273. Strain SM84 was created by transforming the ideR mutant, SM3, with pSM273. Functional IdeR activity was visualized by comple- mentation of SM3s derepression of siderophore synthesis on CAS high-iron plates (Dussurget et al., 1996). A culture of SM84 was grown to stationary phase in 7H9 medium, and cells were harvested and resuspended in buffer A (50mM Tris-HCl, pH7.5, 50mM NaCl, 2mM b-mercaptoethanol) containing a protease inhibitor cocktail (Complete; Boehringer Mannheim). Cells were disrupted by passaging three times by French press at 1200 psi. Cell debris and lipids were removed by 30min ultracentrifugation at 20000r.p.m. Crude extracts were loaded onto a nickel NTA column (Qiagen) and separated by fast protein liquid chromatography (FPLC) using an imidazole gradient of 0100mMin buffer A. IdeR was eluted between 20 and 30mM imidazole. Fractions containing IdeR were pooled and dialysed against 50mM Tris-HCl, pH7.5, 50mM NaCl, 1mM dithiothreitol (DTT) and 1mM EDTA to remove residual nickel. A second FPLC purication was performed using anion exchange on a Source 15Q column (Amersham Pharmacia Biotech) with 50mM Tris-HCl, pH7.5, 50mM NaCl, 1mM DTT and an NaCl gradient of 50500mM. The major IdeR peak eluted between 65 and 85mM NaCl. IdeR was .99% pure as veried by Coomassie staining and Western blotting, with a yield of about 150mg of puried IdeR l 21 culture. Gel retardation assay The gel retardation assay was carried out essentially as described previously (Hamoen et al., 1998; Rodriguez et al., 1999) with minor modications. PCRfragments containing the putative IdeR consensus binding sites were end labelled with T4 polynucleotide kinase and [g- 32 P]-ATP. Binding reactions [20 mM Tris-HCl at pH8, 1 mM DTT, 50 mM KCl, 5mM MgCl 2 , 0.05 mg ml 21 poly-(dI dC), 0.05 mg ml 21 BSA and 10% glycerol] containing <0.1 ng of probe (20000 c.p.m.) were incubated with puried IdeR (0.061 mM) in 20 ml volumes for 30min at room temperature. NiSO 4 (200 mM) was added to binding reactions and, when required, to the acrylamide gels and running buffers. Reactions (15 ml) were loaded without dye to an 8% polyacrylamide gel containing 40 mM Tris acetate at pH8. Gels were run at 110V at room temperature, dried, and bands were visualized by autoradiography. DNase footprint analysis Primers corresponding to either the top or bottom strands of the following promoter regions, mbtAmbtB, mbtI, rv3402c, bfrAbfd, were labelled with T4 polynucleotide kinase and [g- 32 P]-ATP and then used to PCR amplify the corresponding fragments. PCR products were ethanol precipitated and then puried on a 2% agarose gel. Binding reactions with IdeR were performed as described for the gel retardation assay in 20ml with <100000 c.p.m. probe and 1 mM IdeR. Reaction volumes were adjusted to 100ml with a Ca 21 /Mg 21 solution (nal concentrations are 2.5 mM CaCl 2 and 5 mM MgCl 2 ). Then, 0.15U of DNase (Promega) was added, and mixtures were incubated for 1min at room temperature. Reactions were terminated by the addition of 90 ml of stop solution (200 mM NaCl, 20mM EDTA, 1% SDS and 100mgml 21 yeast RNA). Samples were subsequently extracted with phenol chloroform, ethanol precipitated and resuspended in a formamide loading dye. Samples were electrophoresed on a 6% TBE polyacrylamideurea sequencing gel, dried and exposed by autoradiography. MaxamGilbert A1G sequen- cing reactions were performed to identify protected regions. Primer extension analyses RNA was extracted essentially according to the protocol of Manganelli et al. (1999). M. tuberculosis H37Rv was grown as described above in low- and high-iron MM-ADN to late log phase. Bacterial pellets were resuspended in LETS buffer (100 mM LiCl, 10mM EDTA, 10 mM Tris, pH7.8, 1% SDS) and then mixed with an equal volume of phenol chloroform isoamyl alcohol (P:C:IAA; 25:24:1) and broken with a bead beater. Membranes were removed by centrifugation at 14000 r.p.m. for 15 min, and the resulting supernatant was extracted once in P:C:IAA and twice with TRI reagent (Molecular Research Center), according to the manufac- turers protocol. RNA integrity was veried by electrophoretic analysis on a 2% TBE agarose gel. For primer extension, 20 60mg of RNA was hybridized to 1.5 pmol of g- 32 P-labelled 862 B. Gold et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 851865 reverse primer in 1 Carboxydothermus hydrogenoformans reverse transcription buffer (C. therm. kit; Roche) in a nal volume of 10ml. The hybridization programme was 948C for 1.5 min, 648C for 3min, 578C for 5 min, and then the tubes were allowed to cool rapidly to 508C, when they were placed directly on ice. A reverse transcription mixture using the C. therm reverse transcriptase (Roche; nal of 1mM dNTPs, 2.5 mM MgCl 2 , 5% DMSO and 5mM DTT) was added to the annealed primer RNA mixture at a nal volume of 20ml. The cDNA synthesis reaction was at 608C for 1 h followed by 2min at 948C, and then the cDNARNA mixture was ethanol precipitated and washed with 75% EtOH. Samples were resuspended in 6 ml of water and 4 ml of formamide loading buffer and separated on a 6% TBE gel containing 8 M urea at 1800 V. In some cases, a 10 bp ladder (Gibco BRL) was used as a molecular weight standard to determine the length of the primer extension product and, in other cases, a sequencing reaction was performed using the Sequenase 7-deaza-dGTP kit (USB). All transcriptional start points were veried using two primers whose 3 0 ends were 1020 bp apart. Molecular beacon RTPCR (mbRTPCR) To obtain RNA, frozen bacterial pellets were resuspended in 1ml of TRI reagent and 500ml of silica beads and then broken using a bead beater at maximum speed for 21min, with chilling on ice between breaking periods. RNA extraction was performed according to the manufacturers instructions (Molecular Research Center). The mbRTPCR was per- formed essentially as described by Manganelli et al. (1999; 2001), and cDNA was synthesized as described above for primer extension assays. A cocktail of up to ve reverse primers was added per reaction to have all reverse transcriptions (always including sigA) performed in the same tube. RNA (50200ng) was added to each reverse transcrip- tion and, in all cases, a mock reaction lacking reverse transcriptase was performed to obtain copy numbers of DNA contaminating the RNA sample. The PCR reactions typically contained 100800nM of a gene-specic molecular beacon (coupled to tetramethylrhodamine or uorescein) and were performed with the following PCRprogramme: 958Cfor 10min, 10 cycles of 958C for 20s, 658C for 30s and 728C for 30s, and then 35 cycles of 958C for 20s, 608C for 30s and 728C for 30s. Molecular beacons were designed with the aid of the MFOLD server (http://bioinfo.math.rpi.edu/,mfold/dna/form1.cgi) and synthesized as described previously (Tyagi and Kramer, 1996). All molecular beacon and primer sequences are available upon request. Ratios of induction at each time point were solved using the following formula, where X and A are the absolute number of copies of mRNA from geneX or sigA respectively: (X low Fe /A low Fe )/(X high Fe /A high Fe ). Isolation of RNA from THP-1 macrophages infected with M. tuberculosis Mycobacterium tuberculosis infections of THP-1-derived macrophages were performed as described by Manganelli et al. (2000). Briey, THP-1 cells were grown in RPMI-1640 (supplemented with 0.45% glucose, 0.15% sodium pyruvate and 4mM L-glutamine), differentiated into macrophages by 24 h treatment with 50 nM 12-O-tetradecanoylphorbol- 13-acetate (PMA) (Tsuchiya et al., 1982) and plated at a nal concentration of 7.5 10 5 cells ml 21 . To improve long- term macrophage adherence in large volumes, asks were pretreated with 0.2% gelatin overnight at 48C. M. tuberculosis H37Rv were grown in 7H9 to an OD 540 of 0.2 and infected into the THP-1 cells at a multiplicity of infection of 0.51. Extracellular bacteria were removed after 2h by washing twice with phosphate-buffered saline (PBS), and then fresh RPMI containing 50 mgml 21 gentamicin and 20% fetal calf serum (FCS) was added back to the macrophages. At 24 and 72 h, RPMI was removed, macrophages were washed once with RPMI, and then the remaining cells were resuspended in 10 ml of TRI reagent containing polyacryl carrier, frozen immediately on dry ice and then stored at 2808C. At each time point, macrophages were checked for viability using Trypan blue exclusion (Sigma), and viable counts of M. tuberculosis growing intra- and extracellularly were performed in a parallel infection. Expression levels of all genes were normalized against M. tuberculosis that had been exposed to RPMI for 2h or to an M. tuberculosis rolling culture in 7H9. To make RNA, 500ml of silica beads was added to the frozen mix of M. tuberculosis and macrophages, and cells were broken using a bead beater at maximum speed for 21 min, with chilling on ice between breaking periods. RNA extraction was performed with TRI reagent according to the manufacturers instructions (Molecular Research Center). RNA was subjected to DNase treatment (DNA-free kit; Ambion) and then precipitated and resuspended in water treated with diethylpyrocarbonate (DEPC). Ratios of induction at each time point were calculated using the following formula, where X and A are the absolute number of copies of mRNA from geneX or sigA respectively: (X intracellular /A intracellular )/(X extracellular /A extracellular ). Acknowledgements We would like to thank members of the Smith laboratory for support and fruitful discussions. For help with the M. tuberculosis gene expression during macrophage infections, we would like to thank Riccardo Manganelli, Patricia Fontan and Eugenie Dubnau. For excellent advice regarding protein purication, gel shifts and DNase footprints, we would like to thank Leendert Hamoen, Bert Jan Haijema and Kursad Turgay. 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CRISPR/Cas9 Allows Efficient and Complete Knock-In of Adestabilization Domain-Tagged Essential Protein in Ahuman Cell Line, Allowing Rapid Knockdown of Protein Function