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Journal of Bacteriology, January 2006, p. 150-159, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.150-159.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Mycobacterium tuberculosis TrcR Response Regulator Represses Transcription of the Intracellularly Expressed Rv1057 Gene, Encoding a Seven-Bladed ß-Propeller

Shelley E. Haydel^1*, and Josephine E. Clark-Curtiss^1,2,

Departments of Biology,¹ Molecular Microbiology, Washington University, St. Louis, Missouri 63130²

Received 3 August 2005/ Accepted 11 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Mycobacterium tuberculosis TrcR response regulator binds and regulates its own promoter via an AT-rich sequence. Sequences within this AT-rich region determined to be important for TrcR binding were used to search the M. tuberculosis H37Rv genome to identify additional related TrcR binding sites. A similar AT-rich sequence was identified within the intergenic region located upstream of the Rv1057 gene. In the present work, we demonstrate that TrcR binds to a 69-bp AT-rich sequence within the Rv1057 intergenic region and generates specific contacts on the same side of the DNA helix. An M. tuberculosis trcRS deletion mutant, designated STS10, was constructed and used to determine that TrcR functions as a repressor of Rv1057 expression. Additionally, identification of the Rv1057 transcriptional start site suggests that a SigE-regulated promoter also mediates control of Rv1057 expression. Using selective capture of transcribed sequences (SCOTS) analysis as an evaluation of intracellular expression, Rv1057 was shown to be expressed during early M. tuberculosis growth in human macrophages, and the Rv1057 expression profile correlated with a gene that would be repressed by TrcR. Based on structural predictions, motif analyses, and molecular modeling, Rv1057 consists of a series of antiparallel ß-strands which adopt a ß-propeller fold, and it was determined to be the only seven-bladed ß-propeller encoded in the M. tuberculosis genome. These results provide evidence of TrcR response regulator repression of the Rv1057 ß-propeller gene that is expressed during growth of M. tuberculosis within human macrophages.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tuberculosis remains a significant world health burden with approximately one-third of the world's population estimated to be infected and a mortality rate of 2 million people per year (49). Additionally, due to the rapid progression of both tuberculosis and human immunodeficiency virus (HIV) in coinfected individuals, tuberculosis is the leading cause of death in HIV-infected individuals and is the causative agent in 13% of AIDS deaths worldwide (49). Although a live attenuated vaccine and several effective antituberculosis antibiotics are currently available, globally, there are approximately 8 million new cases of tuberculosis per year with 3.2% of these cases represented as multidrug resistant (10). These statistical trends support the need for the development of improved vaccines and vaccination strategies and the identification of therapeutic compounds effective at eliminating active and latent tuberculosis infections.

Mycobacterium tuberculosis is a well-equipped intracellular pathogen, as evidenced by its natural ability to adapt and reside within the human macrophage phagosome. Throughout its life in the human host, M. tuberculosis encounters a range of environments, and as a result, gene regulation must be tightly controlled for bacterial adaptation to occur. Regulation of genes required for M. tuberculosis survival within the human host likely involves coordinated control by two-component signal transduction systems (13). Various hybridization techniques, complete transcriptional profiling using DNA microarrays, and the ability to generate mutants are allowing insights into the gene expression requirements for the M. tuberculosis intracellular lifestyle, and these insights are paramount to understanding mycobacterial gene regulation and adaptive responses to a pathogenic lifestyle within the host.

M. tuberculosis encodes 11 different two-component regulatory systems and several orphan histidine kinase and response regulator genes (5). Although the functions of many of these mycobacterial signal transduction systems remain undefined, recent studies have analyzed the expression profiles of these regulators during M. tuberculosis growth in macrophages (17, 50) and have begun to ascertain the biological role of these regulatory circuits. Mutagenesis of several regulators has established a role for two-component systems in intracellular growth and in vivo survival (35, 36, 38, 42, 50). As a result of various genetic and biochemical analyses, the roles of DevR (DosR) as a regulator of hypoxia-responsive genes and PhoP as a modulator of acylated mannosylated lipoarabinomannans have been discerned (27, 37). Additionally, the consistent trend of autogenous regulation of bacterial two-component systems is evident in M. tuberculosis, with four response regulators, RegX3, TrcR, PrrA, and MprA, capable of binding to their respective promoters and autoregulating expression (12, 16, 19, 20).

Autoregulation of the TrcR response regulator occurs via binding of an AT-rich sequence within the trcR promoter (16). In the studies presented herein, the AT-rich characteristics of the trcR promoter were used to identify the Rv1057 gene which has several AT tracts of DNA within its upstream intergenic sequence. Protein-DNA binding experiments identified two TrcR binding sites within the Rv1057 intergenic sequence, localized a high-affinity 69-bp binding site, and revealed that TrcR generates specific contacts on one side of the DNA helix and wraps around the ends of the DNA binding region. Transcriptional analyses revealed that Rv1057 is repressed by the TrcR response regulator, is expressed during M. tuberculosis growth in human peripheral blood monocyte (PBMC)-derived macrophages, and is likely subjected to control by the extracytoplasmic function (ECF) sigma factor SigE. We present evidence suggesting that Rv1057 belongs to a family of proteins with a repeated domain which adopt a three-dimensional organization known as a ß-propeller (14, 22, 45). Proteins containing the ß-propeller fold are found in many organisms, have been implicated in the pathogenesis of several human diseases, and are diversified in function (14, 26, 39, 45). Although these protein structures are common in eukaryotes, including humans, the ß-propeller fold is considerably less abundant in prokaryotes (14). Pathogenic bacteria containing proteins with ß-propeller domains include the Salmonella enterica serovar Typhimurium and Vibrio cholerae sialidases (neuraminidases) (7, 8), Escherichia coli TolB (40), E. coli YxaL (34), and Gyr/Par DNA gyrases from numerous pathogens (41). Rv1057 represents the only seven-bladed ß-propeller protein encoded in M. tuberculosis, and to our knowledge, this study presents the first evidence of a two-component system controlling expression of a ß-propeller gene.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial media and growth conditions. E. coli was grown in Luria-Bertani (LB) broth or on LB agar plates at 37°C (3). M. tuberculosis was grown at 37°C in Middlebrook 7H9 liquid medium or on Middlebrook 7H9 agar (Difco) supplemented with ADS (bovine serum albumin-fraction V, dextrose, NaCl) enrichment, 0.2% glycerol, and 0.05% Tween 80. Mycobacterium smegmatis was grown at 37°C in Middlebrook 7H9 liquid medium or LB broth supplemented with 0.5% Tween 80 or on LB agar plates. The following concentrations of antibiotics or inducing supplement were added when appropriate: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml for E. coli and 25 µg/ml for mycobacteria; apramycin (Am), 50 µg/ml for E. coli and 30 µg/ml for mycobacteria; hygromycin (Hyg), 150 µg/ml for E. coli and 100 µg/ml for mycobacteria; and isopropyl-ß-D-thiogalactopyranoside (IPTG), 0.1 mM.

Bacterial strains and plasmids. E. coli strain JM109 was used as a host for plasmid constructions and ß-galactosidase analyses. Mycobacterial host strains used for ß-galactosidase analyses included M. tuberculosis H37Rv (wild type), STS10 (trcRS mutant strain), STS35 (H37Rv::pSH489), STS36 (H37Rv::pSH493), STS40 (STS10::pSH489), and STS41 (STS10::pSH493).

The Rv1057 promoter-lacZ fusion was constructed in pSEH100 (16) and the pJEM15 E. coli-mycobacterial shuttle plasmid (47) for ß-galactosidase analyses in E. coli and M. tuberculosis, respectively. Plasmids pSH179 and pSH285 consist of a 633-bp fragment containing 531 bp of the Rv1057 upstream intergenic region and 102 bp of the Rv1057 coding region cloned upstream of the lacZ gene in pSEH100 and pJEM15, respectively. Plasmid pSH33 represents the N-terminal His₆-TrcR recombinant expression plasmid, as previously described (16). Plasmid pSH344 harbors the Rv1057 gene cloned into pGEM-T (Promega Corporation), while plasmid pSH232 harbors the Rv1057 promoter cloned into pGEM-T. The kanamycin (aph) resistance cassette in the pMV306 site-specific integrating mycobacterial vector was replaced with an apramycin (aacC41) resistance cassette, which does not confer kanamycin cross-resistance (6), to create pMV306/Am for generating the trcR- and trcRS-complementing plasmids. The trcR-complementing plasmid, pSH489, consists of 199 bp of the trcR promoter, the trcR coding region, and 14 bp downstream of the trcR stop codon cloned into pMV306/Am. The trcRS-complementing plasmid, pSH493, consists of 199 bp of the trcR promoter, the trcR and trcS coding regions, and 12 bp downstream of the trcS stop codon cloned into pMV306/Am.

Protein purification and treatment of TrcR with acetyl phosphate. The TrcR protein was expressed and purified by nickel-nitrilotriacetic acid agarose columns as previously described (18). In vitro phosphorylation of TrcR by acetyl phosphate was performed as previously described (16, 30). Briefly, TrcR was treated with 30 mM acetyl phosphate in a phosphorylation buffer containing 50 mM Tris-HCl, pH 8, 10 mM MgCl₂, and 3 mM dithiothreitol for 30 min at 37°C. Following phosphorylation with acetyl phosphate, the protein was added to electrophoretic mobility shift assays (EMSAs) (described below).

EMSAs. DNA probes for EMSAs were amplified by PCR and 3' end labeled with digoxigenin (DIG) as described by the DIG gel shift kit manufacturer (Roche). Three Rv1057 intergenic sequence PCR fragments were amplified from M. tuberculosis H37Rv: P_Rv1057, 469 bp, primers Rv1057 F-1 (5'-CACCGACCTCAGCGATCACC-3') and Rv1057 R-2 (5'-GCTGTGCGACGCCGACTGCC-3'); P_Rv1057-1, 281 bp, primers Rv1057 F-1 and Rv1057 R-4 (5'-GTATGTGGCAAACCAGTGCT-3'); and P_Rv1057-2, 208 bp, primers Rv1057 F-3 (5'-AGCACTGGTTTGCCACATAC-3') and Rv1057 R-2. Purified PCR products were quantitated using GeneQuant (Amersham Pharmacia Biotech), and 100 ng of each product was end labeled with DIG-11-ddUTP. EMSA binding reactions contained increasing concentrations of TrcR protein (0, 16, 81, 161, 806, or 1,610 nM) and 0.4 ng of the DIG-labeled Rv1057 promoter PCR fragments (0.07 nM of P_Rv1057, 0.11 nM of P_Rv1057-1, and 0.15 nM of P_Rv1057-2). Electrophoresis and detection of the DNA-protein complexes were performed as previously described (16).

DNase I and hydroxyl radical footprinting analyses. DNase I footprinting analyses were performed as previously described for TrcR (16). For generating the single, end-labeled P_Rv1057 469-bp fragment used in the DNase I analysis, the Rv1057 R-2 primer was end labeled with T4 polynucleotide kinase (Epicenter Technologies) and [-³²P]ATP (specific activity, 6,000 Ci/mmol) and used in a PCR with the Rv1057 F-1 primer and pSH232. Approximately 0.85 nM (65,000 cpm) of the end-labeled P_Rv1057 fragment was used in the DNase I footprinting reactions. For generating the single, end-labeled P_Rv1057-1 281-bp fragment used in hydroxyl radical footprinting analyses, either the Rv1057 F-1 or Rv1057 R-4 primer was end labeled as described above and used in a PCR with M. tuberculosis H37Rv chromosomal DNA. For hydroxyl radical footprinting, labeled P_Rv1057-1 fragments (0.55 to 1.65 nM; 75,000 cpm) were incubated in binding reactions with TrcR (0, 645, or 1290 nM) as previously described (16). For hydroxyl radical cleavage of the TrcR binding reactions, 4 µl each of 0.4 mM iron ammonium sulfate-0.8 mM EDTA, 20 mM sodium ascorbate, and 0.3% (vol/vol) H₂O₂ were mixed and added to the binding reactions. For cleavage of control reactions lacking the TrcR protein, 4 µl each of 0.1 mM iron ammonium sulfate-0.2 mM EDTA, 20 mM sodium ascorbate, and 0.3% (vol/vol) H₂O₂ were mixed and added to the reactions. After a 3-min incubation at room temperature, 10 µl of stop solution (20 mM EDTA, 1% sodium dodecyl sulfate [SDS], 200 mM NaCl) was added with 1 µl of 10-mg/ml glycogen and 150 µl of 95% cold ethanol to terminate the cleavage reaction, and samples were processed as previously described (16). Dideoxynucleotide sequencing reactions were performed using a SequiTherm EXCEL II DNA sequencing kit (Epicenter Technologies) with the same end-labeled primer used in the footprinting reaction, and the products were electrophoresed in parallel with the TrcR footprinting reactions.

Generation of a trcRS mutant strain of M. tuberculosis. The M. tuberculosis trcRS mutant in which the trcRS operon is deleted and replaced with the Hyg resistance cassette was constructed by allelic exchange mutagenesis using the specialized transducing mycobacteriophage system described by Bardarov et al. (3). The trcRS::Hyg allele was constructed by amplifying the flanking regions of the trcRS genes and cloning the fragments on either side of the Hyg resistance cassette. The 931-bp region upstream of the trcRS genes was amplified using primers SH77 (5'-CCCAAGCTTGCTGGACAACGCCAAGCAGG-3') and SH78 (5'-CTAGCTAGCGCGTGTACCCCGACATCGTC-3'), which contain HindIII and NheI sites (underlined) at their respective 5' termini. The 1,026-bp region downstream of the trcRS genes was amplified using primers SH79 (5'-TGCTCTAGAGCCAGACGGTGTTTCGGGTG-3') and SH80 (5'-CGGGGTACCCCGACCCCACCAAGCTCATC-3'), which contain XbaI and KpnI sites (underlined) at their respective 5' termini. The PCR products were cloned into pYUB854 (3), flanking the Hyg cassette, to create pSH268. The ligation mixture of PacI-digested pSH268 and PacI-digested concatemerized phAE87 was packaged using GigaPack III packaging extracts (Stratagene) and transduced into E. coli HB101. Phasmid DNA was prepared from pooled Hyg-resistant transductants, restriction enzyme-digested to verify the presence of the desired insert, electroporated into M. smegmatis mc²155, and plated for mycobacteriophage plaques at 30°C. A high-titer mycobacteriophage stock was generated from a confirmed temperature-sensitive phage plaque and was used to infect M. tuberculosis H37Rv as previously described (3). Hygromycin-resistant colonies were picked after 3 to 4 weeks of growth at 37°C and screened by PCR for deletion of the trcRS genes. Southern blot analysis of candidate clones confirmed the trcRS deletion and the presence of the Hyg resistance cassette. One of the confirmed trcRS::Hyg deletion mutants, designated M. tuberculosis STS10, was used in subsequent experiments.

Analysis of ß-galactosidase activity. TrcR regulatory experiments were performed in E. coli with the Rv1057 promoter-lacZ fusion plasmid, pSH179, and recombinant His₆-TrcR produced from the IPTG-inducible expression plasmid, pSH33, as previously described (16). Plasmids pSH179 and pSH33 were electrotransformed into E. coli JM109. Overnight cultures of positive transformants were grown, diluted in fresh LB medium, and grown to an optical density at 600 nm (OD₆₀₀) between 0.3 and 0.5 before induction with 0.1 mM IPTG for 60 min. ß-Galactosidase measurements were performed as previously described (16) and expressed as Miller units (32).

Cultures of M. tuberculosis H37Rv, STS10, STS35, STS36, STS40, or STS41 harboring pJEM15 or the Rv1057 promoter-lacZ fusion plasmid, pSH285, were grown in 7H9 medium with the appropriate antibiotics at 37°C. Cells were collected at an optical density at 600 nm of 0.5 to 0.8, washed with cold phosphate-buffered saline, and resuspended in 10 mM Tris-HCl, pH 8.0. Cell suspensions were transferred to 2-ml screw-cap tubes containing 0.5 ml of 0.1-mm-diameter zirconia/silica beads (Biospec Products) and were subjected to three 45-s pulses in a Fast Prep homogenizer (Qbiogene, Inc.) with a 1-min rest on ice between pulses. Protein concentrations of the cell extracts were determined by using the Bio-Rad protein assay, with bovine serum albumin as the standard. ß-Galactosidase activity for the mycobacterial extracts was performed as described by Miller (32). Units of ß-galactosidase specific activity were determined using the formula OD₄₂₀ x 380/min at 28°C x mg protein, and they were expressed as nanomoles of nitrophenol produced per minute per milligram of protein (32).

Primer extension. Total RNA was isolated from M. tuberculosis H37Rv by mechanical lysis with 0.1-mm-diameter zirconia/silica beads and TRIzol reagent (Invitrogen) in a Fast Prep homogenizer. Total RNA was then subjected to purification with RNeasy columns and on-column DNase I treatment (QIAGEN). A primer that anneals 71 nucleotides (nt) downstream of the Rv1057 translational start site (Rv1057 GSP1, 5'-CGGAATCTTGACCACGGCGGAGCCC-3') was end labeled by incubation with [-³²P]ATP and T4 polynucleotide kinase and used in a reverse transcription reaction with 5 µg of M. tuberculosis total RNA and Superscript III reverse transcriptase (Invitrogen). Primer extension reactions were repeated with three independently derived RNA samples, and similar results were obtained. Dideoxynucleotide sequencing reactions were performed using a SequiTherm EXCEL II DNA sequencing kit (Epicenter Technologies) with the same end-labeled primer used in the Rv1057 reverse transcriptase reaction, and the products were electrophoresed in parallel with the Rv1057 primer extension reaction.

Expression analysis using M. tuberculosis SCOTS probes. Plasmids pSH344, harboring the M. tuberculosis Rv1057 gene cloned into the pGEM-T vector (Promega Corporation), pSH33 (18), and pSH337 (16) were digested with the appropriate restriction enzymes to release the Rv1057, trcR, and clpC inserts, respectively, separated by agarose gel electrophoresis, and analyzed by Southern blotting using the selective capture of transcribed sequences (SCOTS) cDNA probes as previously described (15, 16). The M. tuberculosis SCOTS cDNA probes were developed from mRNAs specifically expressed during intracellular growth within human PBMC-derived macrophages for 18, 48, or 110 h (15). Briefly, total RNA was isolated from M. tuberculosis-infected human macrophages and converted to cDNA with terminal linker sequences added by reverse transcription. The cDNA was captured by hybridization with biotinylated H37Rv chromosomal DNA that had been prehybridized with rrnA DNA. Prehybridization with rrnA DNA allows for an increased number of cDNA molecules derived from mRNA to be detected (21). The cDNA-chromosomal DNA hybrids were then captured with streptavidin-coated magnetic beads and denatured, and the cDNAs were amplified by PCR. Three rounds of selective capture of cDNAs were performed before preparing DIG-labeled probes from each cDNA mixture. DIG-labeled SCOTS probes were generated by PCR or random-primed labeling with DIG-dUTP as described by the manufacturer (Roche). Three independent hybridizations were performed with each SCOTS cDNA probe to verify reproducibility of the analyses, and similar results were obtained.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Genomic search for AT-rich sequences similar to the trcR promoter. The trcR promoter contains a 28-bp stretch of AT-rich DNA that is involved in TrcR binding (16). To determine if segments of this trcR promoter AT tract of DNA were present elsewhere in the M. tuberculosis chromosome, we searched both strands of the M. tuberculosis H37Rv genome for exact matches to different portions (5'-GCGACATTT-3', 5'-ATTTGAA-3', 5'-GAAAAATTT-3', and 5'-AAATTTA-3') of the trcR AT-rich region. These searches revealed 98 identified regions within the H37Rv genome that contained at least one exact match of the search oligonucleotides (data not shown). One of the identified sequence matches (5'-ATTTGAA-3') was located within the intergenic region located upstream of the Rv1057 gene (see Fig. 2C). In addition, the Rv1057 intergenic region exhibited several extended stretches of AT-rich regions containing search oligonucleotide sequences with one nucleotide mismatch (5'-GTTTGAA-3', 5'-AAATATA-3', and 5'-AAATTTT-3').

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FIG. 2. TrcR footprinting analysis of the Rv1057 intergenic sequence. Binding reaction mixtures containing the 469-bp (A) or 281-bp (B) end-labeled Rv1057 sequence were subjected to DNase I digestion (A) or hydroxyl radical cleavage (B) in the absence of TrcR (–) or with increasing concentrations (nM) of TrcR. Lanes G, A, T, and C represent DNA sequencing reactions of Rv1057 using the same labeled Rv1057 primer used in the corresponding footprinting reactions. (C) Double-stranded DNA sequence of the Rv1057 upstream intergenic sequence. The DNase I protected region is shown with a thick line, and the hydroxyl radical protected bases are indicated with several thin lines located above and below the sequences. The TrcR binding candidate sequence match identified in genomic searches is bolded. Lowercase letters denote the translational start site, and the primer annealing sites are shown with arrows.

TrcR binds to AT-rich sequences upstream of Rv1057. To determine if TrcR interacts with the regulatory region of Rv1057, a 469-bp fragment (–62 through –530, in relation to the Rv1057 translational start site) containing the identified candidate search sequence and the accompanying AT-rich region was amplified, purified, and end labeled (as described in Materials and Methods) (Fig. 1A). EMSAs with the 469-bp labeled P_Rv1057 PCR product revealed that TrcR, either in its in vitro phosphorylated (TrcRP) or unphosphorylated state, induced gel shifts of the Rv1057 upstream sequence in a concentration-dependent manner (Fig. 1B). It should be noted that although there is no experimental evidence to support in vitro TrcR phosphorylation by acetyl phosphate, an increased binding affinity of the P_Rv1057 fragment is detected in the TrcRP EMSAs (Fig. 1B). As a control for the specificity of TrcR binding to Rv1057, binding reactions with a 50-fold excess of unlabeled P_Rv1057 DNA as a specific competitor resulted in a loss of TrcR binding to the labeled P_Rv1057 DNA (Fig. 1B). However, with the addition of a 50-fold excess of nonspecific poly d(I-C) DNA, specific binding activity of TrcR to the P_Rv1057 fragment was retained (Fig. 1B).

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FIG. 1. EMSAs demonstrating TrcR binding to the Rv1057 promoter. (A) Schematic representation of Rv1057 and the upstream adjacent region. The Rv1057 upstream intergenic regions used in the EMSAs are shown as shaded rectangles and are represented by a 469-bp DNA fragment, PRv1057 (panel B), a 281-bp DNA distal fragment, PRv1057-1 (panel C), and a 208-bp DNA proximal fragment, PRv1057-2 (panel D). The Rv1057 promoter fragments are shown unbound (DNA only), with increasing amounts of TrcR, and with increasing amounts of TrcRP. The concentration of TrcR (nM) is indicated above each lane. The positions of the free DNA (F) and complexed DNA (C) are shown with arrows.

Since the 469-bp Rv1057 fragment harbors several AT-rich tracts of DNA and the EMSAs showed smearing or multiple gel shift products (Fig. 1B), additional EMSAs were performed with distal and proximal portions of this sequence to determine if TrcR was binding to more than one site. A 281-bp distal fragment of the Rv1057 upstream sequence, P_Rv1057-1, was bound by TrcR or TrcRP with only a small amount of labeled DNA remaining unbound upon incubation with increasing concentrations of TrcR or TrcRP (Fig. 1C). In contrast, TrcR binding of the proximal 208-bp P_Rv1057-2 upstream sequence was minimal with increasing concentrations of protein, and the TrcR-induced DNA shift was smaller than those for the 469-bp and 281-bp Rv1057-TrcR EMSAs (Fig. 1D). These data reveal that TrcR binds with different affinities to at least two sites within the Rv1057 upstream sequence. However, the different binding affinities of the two smaller Rv1057 fragments do not discount the possibility that cooperative binding of both sites is important.

To determine the precise region of TrcR binding within the Rv1057 regulatory region, DNase I and hydroxyl radical footprinting analyses were performed with increasing concentrations of TrcR (Fig. 2). Using the end-labeled 469-bp P_Rv1057 fragment (Fig. 1A) in DNase I footprinting reactions, TrcR protected a 69-bp AT-rich sequence located 347 bp upstream of the Rv1057 translational start site (Fig. 2A and C). The 69-bp TrcR protected sequence is located within the TrcR-bound 281-bp P_Rv1057-1 distally located upstream sequence identified in the EMSAs (Fig. 1A and C). Despite repeated efforts, slight TrcR protection of sequences located within the 208-bp P_Rv1057-2 proximally located sequence was not evident in the TrcR footprinting (DNase I or hydroxyl radical) reactions. Since AT tracts of DNA are poorly cleaved by DNase I (9), both strands of the 281-bp P_Rv1057-1 upstream sequence (Fig. 1A) were subjected to hydroxyl radical footprinting (Fig. 2B). Upon incubation with increasing concentrations of TrcR, seven tracts of the Rv1057 sequence ranging from 4 to 7 nucleotides were specifically contacted and protected by TrcR from hydroxyl radical cleavage (Fig. 2B). At both ends of the protected sequence, TrcR appears to wrap around the DNA, as shown by the same sequences protected by hydroxyl radical cleavage (Fig. 2C). However, the internal contacted sequences occur at regular, phased intervals at approximately every 11 bp with the complementary strands exhibiting the same pattern offset by two or three bases, indicating that TrcR is interacting with Rv1057 on the same side of the DNA helix (Fig. 2C).

Transcriptional analysis of the Rv1057 promoter-lacZ fusion in E. coli. To study the role of TrcR in Rv1057 transcription, an Rv1057 promoter-lacZ fusion was constructed in pSEH100 (16), and ß-galactosidase activity was analyzed in the presence and absence of TrcR expression in E. coli. In E. coli cells harboring the Rv1057 promoter-lacZ plasmid and the recombinant TrcR expression plasmid, a decrease in promoter activity was shown by a reduction in ß-galactosidase activity (Fig. 3). After 60 min of TrcR induction, there was a 4.4-fold decrease in ß-galactosidase activity from the Rv1057 promoter (Fig. 3). In addition, a greater than fourfold reduction in Rv1057 promoter activity was evident after the first 15 min of TrcR induction, indicating a sharp reduction in expression in the presence of increased amounts of TrcR (data not shown). These results indicate that the TrcR response regulator negatively regulates Rv1057 transcription in E. coli.

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FIG. 3. Regulation of the Rv1057 promoter-lacZ fusion in the presence of TrcR in E. coli. The Rv1057 promoter-lacZ fusion plasmid, pSH179, was introduced into E. coli with and without the inducible TrcR expression plasmid, pSH33. Column 1, Rv1057 promoter-lacZ plasmid (pSH179); column 2, Rv1057 promoter-lacZ plasmid (pSH179) and uninduced TrcR (pSH33); column 3, Rv1057 promoter-lacZ plasmid (pSH179) and TrcR (pSH33) induced with 0.1 mM IPTG for 60 min. ß-Galactosidase activities are expressed as Miller units. Results are the averages and standard errors from at least three independent experiments.

Construction of an M. tuberculosis trcRS mutant. The trcRS two-component regulatory system genes are adjacent on the M. tuberculosis chromosome with 7 bp of intervening sequence separating the two open reading frames (Fig. 4A) (18). Using the specialized transducing mycobacteriophage system (3), an M. tuberculosis trcRS mutant was constructed. In this mutant, the hygromycin resistance cassette flanked by res sites replaced >96% of the trcRS coding sequences, leaving only the first 25 bp of trcR and the last 62 bp of trcS (Fig. 4A). Generation of the trcRS mutant, designated M. tuberculosis STS10, was confirmed by Southern blot analysis using the trcS gene or the hygromycin cassette as probes (Fig. 4B). Growth characteristics of the mutant and wild-type strains were analyzed by observing colony morphologies and by measuring the absorbance of the cultures during logarithmic and stationary phases of growth. No significant differences in the growth rate were detected when cultures were grown in supplemented Middlebrook 7H9 liquid media, and no differences in colony morphology were observed when the wild-type and mutant strains were grown on Middlebrook 7H10 agar (data not shown).

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FIG. 4. Construction of an M. tuberculosis trcRS deletion mutant by allelic replacement. (A) Schematic representation of the chromosomal organization of wild-type M. tuberculosis, H37Rv, and the trcRS mutant, STS10, which has been disrupted by a hygromycin resistance cassette. The shaded bars indicate DNA probes used for Southern blot analysis. (B) Genomic DNA was digested with PvuII and hybridized with the trcS gene or the hygromycin resistance cassette. Molecular size markers (in kilobases) are shown on the left.

The trcRS two-component system represses Rv1057 transcription in M. tuberculosis. To assess the ability of the trcRS system to repress Rv1057 in M. tuberculosis, as was evident in E. coli, an Rv1057 promoter-lacZ fusion plasmid, pSH285, was introduced into the H37Rv wild-type strain and the STS10 trcRS mutant derivative, and levels of ß-galactosidase production were compared. As shown in Table 1, low levels of Rv1057 promoter-driven ß-galactosidase activity were detected in wild-type H37Rv. However, in the STS10 trcRS mutant, there was a 29-fold increase in Rv1057 expression compared to the level of expression in H37Rv (Table 1). Low background levels of ß-galactosidase activity from the pJEM15 vector in H37Rv and STS10 confirmed that the detected transcriptional differences were due to the Rv1057 promoter (Table 1). The significant increase in the Rv1057-induced ß-galactosidase activity in STS10 indicates that the trcRS system is indeed able to repress Rv1057 expression in M. tuberculosis. These results combined with TrcR repression of Rv1057 transcription in E. coli and the ability of TrcR to bind to the Rv1057 promoter clearly demonstrate that the trcRS two-component system negatively regulates Rv1057.

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TABLE 1. Expression analysis of Rv1057 in M. tuberculosis H37Rv and STS10

To provide further support that the increase in the level of Rv1057 expression in STS10 was due to the absence of the trcRS two-component system, genetic complementation experiments with trcRS were performed. Since TrcR was able to bind to the Rv1057 promoter and repress Rv1057 expression in E. coli, the ability of the trcR response regulator to complement the trcRS mutation without the trcS histidine kinase was also assessed. For single-copy genetic complementation experiments, site-specific integrating plasmids pSH489 or pSH493 containing either the trcR gene or the trcRS locus, respectively, were independently introduced and integrated into the wild-type H37Rv and trcRS mutant STS10 strains. For trcRS or trcR complementation analysis of Rv1057 transcription, the Rv1057 promoter-lacZ plasmid, pSH285, or the pJEM15 vector was introduced into strains listed in Table 1. In strains STS10::trcRS and STS10::trcR harboring the pSH285 plasmid, complementation of the trcRS mutation with either trcRS or trcR was evident by complete restoration of Rv1057 repression to wild-type levels (Table 1). As suggested by the ability of TrcR to repress Rv1057 transcription in E. coli, the trcR response regulator without its cognate trcS histidine kinase was able to fully complement the trcRS mutation in STS10 by repressing the Rv1057 promoter and preventing ß-galactosidase synthesis (Table 1).

Identification of the SigE-regulated Rv1057 promoter. Having determined that TrcR represses expression of Rv1057 by binding to an AT-rich sequence located 347 bp upstream of the ATG start site, we sought to identify the Rv1057 transcriptional initiation site and promoter region. A 185-bp primer extension product was identified (Fig. 5A) that localizes the Rv1057 transcriptional start site to an adenine nucleotide located 134 nt upstream of the ATG start codon (Fig. 5B). As shown in Fig. 5B, a highly conserved core Shine-Dalgarno sequence, GGAGG, is located 8 nt upstream of the ATG initiation codon, and putative promoter sequences of –10 and –35 were identified upstream of the Rv1057 transcriptional start site. The Rv1057 –10 sequence of GGTTG is identical to the consensus sequence (G/C)GTTG of M. tuberculosis ECF sigma factor SigE-recognized promoters (29), while the –35 sequence of GGGCA is identical in 4 of 5 nt to the consensus sequence GG(A/G)(A/C)C of ^E promoters. These results, suggesting that Rv1057 is regulated by ^E, are consistent with a previous study which identified Rv1057 as a ^E-regulated gene upon exposure to SDS stress and as having a putative SigE sigma factor promoter (29).

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FIG. 5. (A) Primer extension analysis of the Rv1057 transcript showing the transcriptional start site. Primer extension was performed using 5 µg of M. tuberculosis total RNA and the labeled Rv1057 GSP1 primer. Lanes G, A, T, and C represent DNA sequencing reactions of Rv1057 using the labeled Rv1057 GSP1 primer. (B) Nucleotide sequence of the Rv1057 promoter region. The Rv1057 transcriptional initiation site (+1) is shown with a bent arrow indicating the direction of transcription. The Rv1057 ATG start codon and open reading frame are shown in lowercase. The –10 and –35 sequences are underlined, and exact matches to the mycobacterial E consensus sequence are bolded. The core Shine-Dalgarno sequence is double underlined, and the Rv1057 GSP1 primer annealing site is shown with an arrow.

SCOTS analysis of Rv1057 expression in M. tuberculosis-infected human PBMC-derived macrophages. The SCOTS methodology was developed as a method for analyzing bacterial gene expression during M. tuberculosis growth in human PBMC-derived macrophages (15). To determine how the expression profile of Rv1057 compares with trcR when M. tuberculosis is growing intracellularly, cDNA SCOTS probes generated after 18 h, 48 h, and 110 h of growth in human PBMC-derived macrophages (15) were used in Southern hybridizations. As previously described (16), SCOTS analysis revealed very weak expression of the trcR response regulator gene after 18 h of intracellular growth (Fig. 6). The Rv1057 gene exhibits low levels of expression at 18 h, significantly stronger expression at 48 h, and no detectable expression after 110 h of growth in infected human macrophages (Fig. 6). The increased expression of Rv1057 during early growth in macrophages correlates with a gene that would be repressed by TrcR since trcR expression decreases with continued growth in macrophages. Although this correlation is evident at 18 h and 48 h, the lack of detection of trcR and Rv1057 at 110 h could indicate the existence of additional factors regulating expression of Rv1057. Nevertheless, the detection of gene expression after M. tuberculosis infection of human macrophages suggests that Rv1057 is important for early intracellular growth.

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FIG. 6. Detection of Rv1057 expression during M. tuberculosis intracellular growth in human PBMC-derived macrophages. M. tuberculosis SCOTS cDNA probes (15) generated after 18 h, 48 h, and 110 h of growth in macrophages were used in Southern blot analysis of Rv1057, trcR, and clpC genes. Expression of the Rv1057 and trcR genes are indicated with single and double asterisks, respectively, and the clpC fragment is used as a hybridization control (16).

Rv1057 encodes a seven-bladed ß-propeller protein. The Rv1057 gene encodes a 40.7-kDa protein that is described by Cole et al. (5) as a conserved hypothetical protein. A BLAST search (1) revealed that the Rv1057 protein exhibits significant similarity to numerous Methanosarcina surface layer proteins (SLPs). In searching for structural homology or conserved motifs using position-specific sequence similarities, iterative PSI-BLAST searches (1) revealed that Rv1057 exhibits structural homology with a Methanosarcina mazei YVTN (Tyr-Val-Thr-Asn) repeat SLP (24), the Podospora anserina WD (Trp-Asp) repeat ß-transducin-like protein HET-D2Y (11), and a Synechocystis WD repeat ß-transducin homolog with significance by iterations one (E = 3 x 10^–16), two (E = 4 x 10^–57), and three (E = 1 x 10^–63), respectively. The crystal structure of the M. mazei SLP protein reveals the presence of an N-terminal domain that displays a highly symmetrical, seven-bladed ß-propeller fold (24). The conserved WD repeats in the ß-subunit of G-proteins are also organized as a seven-bladed ß-propeller structure extending outward from a central pore (25, 46). The structure-based alignment of the M. tuberculosis Rv1057 protein and the M. mazei SLP seven ß sheets that form the blades of the ß-propeller fold is shown in Fig. 7. The ß-propeller folds often exhibit circular permutations whereby sequence repeats may not coincide with structural repeats (43). In addition, sequence repeats of 40 to 50 residues can be interrupted by loop insertions that may extend the repeat length and hinder fold recognition (22). As depicted in the structural-based alignment (Fig. 7), the Rv1057 ß-propeller exhibits circular permutation and an extended loop insertion in blade 4. Stabilization of the ß-propeller fold is usually accomplished by ring closures that involve tethering of the N and C termini into one modular ß sheet. The closure utilizes hydrophobic interactions between the ß sheets and is referred to as "Velcro" closing (33). Based on conserved ß-propeller motifs and molecular modeling, circular closure of the Rv1057 ß-propeller is achieved by a 1 + 3 "Velcro" closure with a combination of ß-strands from the N- and C-terminal protein ends to form the seventh blade (Fig. 7).

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FIG. 7. ß-Propeller blade sequence structure-based alignment of the M. tuberculosis Rv1057 protein and the M. mazei SLP (protein data bank entry code 1L0Q) (24) containing AXSPD and YVTN domain motifs. The alignment was generated using hidden Markov models, was adjusted to include circular permutations of the sequence with respect to the structural repeats (43), and is represented using the Boxshade server (http://www.ch.embnet.org/software/BOX_form.html) and default parameters. Highly conserved residues (seven or more) and conserved or similar residues are highlighted in black and gray, respectively. Amino acid residues representing each sequence repeat are shown to the right of the alignment. The four predicted Rv1057 ß strands and the ß strands identified in the crystal structure of M. mazei SLP (24) are shown above the alignment. The N and C termini of each domain are adjacent in the last blade of the ß-propeller and are separated by two vertical lines. An extended loop insertion of 28 residues in blade 4 of the Rv1057 ß-propeller structure is omitted and indicated in parentheses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
M. tuberculosis is a facultative intracellular pathogen that is capable of residing within the phagosomal compartment of human macrophages. Current research is oriented toward identification of M. tuberculosis genes that are expressed during growth in macrophages. These intracellularly expressed genes are likely to be important in M. tuberculosis pathogenesis. However, in regard to regulatory systems, absence of detectable expression does not necessarily abrogate importance. During M. tuberculosis growth in human PBMC-derived macrophages, the trcR and trcS genes are expressed at low levels during early stages, but not after 48 or 110 h, of continued intracellular M. tuberculosis growth (Fig. 6) (16). Low levels of intracellular expression followed by the lack of expression of the trcR-trcS system could suggest that trcRS regulatory control is not important during M. tuberculosis human macrophage growth and survival. However, in broth-grown cultures, the trcR response regulator represses the Rv1057 gene which is expressed during M. tuberculosis intracellular growth. Thus, derepression of Rv1057 and other genes in the trcRS regulon upon M. tuberculosis entry into human macrophages could be an important facet in the trcRS regulatory control.

Schnappinger et al. (44) performed transcriptional profiling of intraphagosomal M. tuberculosis compared to growth of M. tuberculosis in broth and determined that Rv1057 expression is induced during intracellular growth in resting and gamma interferon-activated macrophages isolated from wild-type and nitric oxide synthase 2-deficient mice. Although expression of trcR and trcS was not significantly induced during intracellular growth, slight induction profiles were evident (44). These transcriptional profiling data during intracellular growth correlate with the SCOTS human macrophage expression data whereby Rv1057 expression is elevated compared to trcR expression and also support the putative role of TrcR derepression of Rv1057 during intracellular growth.

Identification of the Rv1057 transcriptional initiation site and subsequent analysis of the upstream region revealed the presence of an ECF sigma factor ^E promoter (Fig. 5). The M. tuberculosis sigE gene is induced in human macrophages (2, 15, 23) and upon exposure to environmental stresses including heat shock, SDS-induced surface stress, and oxidative stress (23, 29). The ^E sigma factor clearly is involved in M. tuberculosis pathogenesis as a sigE mutant exhibits reduced growth in human and murine macrophages and is severely attenuated in both immunocompetent and immunodeficient mice (2, 28). As a likely member of the ^E global regulon involved in environmental stress response and virulence (29), the TrcR-repressed Rv1057 gene could play a role in mediating M. tuberculosis pathogenesis. The hypothesis that M. tuberculosis utilizes fatty acids as carbon sources during in vivo growth and persistence (31) suggests that genes involved in the metabolism of fatty acid substrates are important in the mycobacterial infection process. In addition to several genes involved in cellular and lipid metabolism and fatty acid degradation, including members of the ^E regulon (29), Rv1057 is induced upon M. tuberculosis growth in the presence of free fatty acid as the sole carbon source (44), suggesting a possible role in intraphagosomal survival and metabolism of lipid substrates.

Repression of Rv1057 by TrcR was demonstrated in both E. coli and M. tuberculosis laboratory-grown cultures. Expression of the recombinant TrcR protein was able to repress transcription of Rv1057 in E. coli, while the absence of the trcRS system in a mutant M. tuberculosis strain resulted in activation of Rv1057 transcription. From our studies of Rv1057 transcription in wild-type and trcRS mutant M. tuberculosis, the trcRS mutation is fully complemented by either the trcR response regulator or the complete trcRS two-component system. These results indicate that (i) TrcS communication with TrcR may not be important for repression of Rv1057, (ii) TrcR communicates with another histidine kinase, (iii) TrcR is phosphorylated by small phosphodonor molecules, or (iv) TrcR phosphorylation is not absolutely required for repression of Rv1057.

Although it is currently unclear how TrcR represses Rv1057, two models of repression can be hypothesized. One model involves the possible preclusion of RNA polymerase binding due to a low-affinity TrcR binding site. As shown in Fig. 1, TrcR binds to at least two sites within the Rv1057 upstream region. However, the binding affinities vary greatly with TrcR exhibiting stronger affinity for the distally located sequence and weaker affinity for a proximally located sequence (Fig. 1C and D). Despite the inability to localize the low-affinity TrcR binding site in footprinting analyses, the ability of TrcR to bind within the proximal region of the Rv1057 upstream sequence could interrupt RNA polymerase binding and facilitate Rv1057 repression. Another possible model involves the intrinsic DNA curvature of the Rv1057 intergenic sequence. A computer-generated analysis predicted that there are three regions of DNA curvature within the Rv1057 intergenic sequence (data not shown). Based on the Trifonov model (4, 48), the AT tract (AAATTTTT) located 124 bp upstream of the Rv1057 transcriptional start site and 129 bp downstream of the TrcR binding region exhibits a significant degree of curvature (data not shown). Two additional regions of predicted DNA curvature were centered around the sequences TAAAA and TCATTTT located 252 bp and 326 bp, respectively, upstream of the Rv1057 transcriptional start site (data not shown). The presence of two intrinsic regions of DNA curvature located between the Rv1057 transcriptional initiation site and the TrcR binding region could facilitate DNA bending leading to TrcR-RNA polymerase interactions that would prevent transcription. While the Rv1057 intergenic sequence is predicted to have multiple regions of intrinsic DNA curvature, circular permutation studies with TrcR did not reveal protein-induced bending of the Rv1057 DNA (data not shown).

As determined by sequence and predicted structural analyses, the Rv1057 protein encodes a 40.7-kDa seven-bladed ß-propeller protein with a repetitive AXSPD motif similar to that of TolB (40) and a loosely conserved YVTN motif similar to the M. mazei SLP (24) (Fig. 7). Despite similar topology and structure, ß-propeller proteins are extremely diversified with functions including enzyme catalysis, signal transduction, ligand binding, transport, mediation of protein-protein interactions, control of cell division, and modulation of gene expression (14, 22, 45). Similar to other bacteria, M. tuberculosis appears to encode few ß-propeller proteins, and pattern searches revealed the presence of four ß-propeller proteins in addition to the Rv1057 seven-bladed ß-propeller. Although the functions of ß-propellers are diverse, assessing their regulation and determining the high resolution structure of the Rv1057 protein and the additional four ß-propellers will enhance the ability to determine the roles and importance of these proteins in M. tuberculosis pathogenesis.

 
We gratefully acknowledge the late Stoyan Bardarov for providing phAE87 and pYUB854 and for valuable guidance and technical expertise related to the mycobacteriophage mutagenesis procedure. We are grateful to Chris Ponting for performing the Rv1057 ß-propeller structural alignment. We thank Roy Curtiss III for critical review of the manuscript and for helpful suggestions and discussions.

This research was supported by a Heiser Program of the New York Community Trust fellowship (to S.E.H.), NIH training grant fellowship AI07172 (to S.E.H.), and NIH grant AI46428 (to J.E.C.-C.).

 
* Corresponding author. Present address: Center for Infectious Diseases and Vaccinology, The Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ 85287. Phone: (480) 727-7234. Fax: (480) 965-6899. E-mail: Shelley.Haydel{at}asu.edu.

Present address: Center for Infectious Diseases and Vaccinology, The Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ 85287.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, January 2006, p. 150-159, Vol. 188, No. 1
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