Mycobacterium tuberculosis mtrA merodiploid strains with point mutations in the signal-receiving domain of MtrA exhibit growth defects in nutrient broth

doi:10.1016/j.plasmid.2011.01.002

Plasmid. Author manuscript; available in PMC 2012 May 1.

Published in final edited form as:

Plasmid. 2011 May; 65(3): 210–218.

Published online 2011 February 3. doi: 10.1016/j.plasmid.2011.01.002

PMCID: PMC3076548

NIHMSID: NIHMS270783

Mycobacterium tuberculosis mtrA merodiploid strains with point mutations in the signal-receiving domain of MtrA exhibit growth defects in nutrient broth

Maha Al Zayer,^¶ Dorota Stankowska,^¶ Renata Dziedzic, Krishna Sarva, Murty V. Madiraju, and Malini Rajagopalan^*

Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, TX- 75708

^* Address for Correspondence: Email: murty.madiraju/at/uthct.edu

^¶Equal Contribution

The publisher's final edited version of this article is available at Plasmid

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Abstract

The genetic and biochemical aspects of the essential Mycobacterium tuberculosis MtrAB two-component regulatory signal transduction (2CRS) system have not been extensively investigated. We show by bacterial two-hybrid assay that the response regulator (RR) MtrA and the sensor kinase MtrB interact. We further demonstrate that divalent metal ions [Mg(2+), Ca(2+) or both] promote MtrB kinase autophosphorylation activity, but only Mg(2+) promotes phosphotransfer to MtrA. Replacement of the conserved aspartic acid residues at positions 13 and 56 with alanine (D13A), glutamine (D56E) or asparagine (D56N) prevented MtrA phosphorylation, indicating that these residues are important for phosphorylation. The MtrA_D56E and MtrA_D13A proteins bound to the promoter of fbpB, the gene encoding antigen 85B protein, efficiently in the absence of phosphorylation, whereas MtrA_D56N did not. We also show that M. tuberculosis mtrA merodiploids overproducing MtrA_D13A, unlike cells overproducing wild-type MtrA, grow poorly in nutrient broth and show reduced expression of fbpB. These latter findings are reminiscent of a phenotype associated with MtrA overproduction during intramacrophage growth. Our results suggest that MtrA_D13A behaves like a constitutively active response regulator and that further characterization of mtrA merodiploid strains will provide valuable clues to the MtrAB system.

Keywords: Mycobacterium tuberculosis, response regulator, signal transduction, mutants, merodiploids, real-time PCR, EMSA

1. Introduction

The survival of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon infection is believed to depend, in part, on the activity of several regulatory networks, one of which is a paired histidine-aspartate two-component regulatory system (2CRS). The 2CRS includes a sensor histidine kinase (which is a membrane-bound protein) and a response regulator protein (which is often a cytosolic protein) [reviewed (Hoch, 2000; Stock et al., 2000)]. Upon detecting an environmental signal, sensor kinases undergo autophosphorylation by transferring the g-phosphoryl group from ATP to a highly conserved histidine residue located in its transmitter domain. The phosphoryl group is then transferred to the regulatory domain of the cognate RR by a phospho-relay process. This, in turn, results in the activation of the output domain of the RR, which usually functions as a transcriptional regulator [reviewed in (Hoch, 2000; Stock et al., 2000)]. Phosphorylation of an RR often leads to its dimerization, although some RRs such as CheA remain monomeric.

The M. tuberculosis genome contains 11 paired 2CRSs; however, MtrAB is the only essential 2CRS (Cole et al., 1998; Via et al., 1996; Zahrt and Deretic, 2000). The mtrB gene of M. avium is implicated in the regulation of cell wall permeability and the colony-switch phenotype (Cangelosi et al., 2006). The MtrAB system of Corynebacterium glutamicum is implicated in the osmo-stress response (Moker et al., 2004; Moker et al., 2007). Recent studies indicate that elevated intracellular levels of phosphorylation-competent MtrA are detrimental for M. tuberculosis growth in macrophages (Mµ) (Fol et al., 2006), whereas elevated levels of mtrB along with mtrA reverse the growth defect, indicating that one role of MtrB kinase is to help regulate the phosphorylation status of MtrA. Presumably, it is the ratio of MtrA to MtrA~P, rather than their absolute levels, that is critical for optimal proliferation. These studies also revealed that the dnaA promoter is MtrA target (Fol et al., 2006). The above studies assumed that MtrB kinase phosphorylates MtrA and that signals for MtrA phosphorylation are enriched in the intracellular growth environment. Other recent studies identified MtrA motifs in the replication origin (or oriC) and the promoter for Ag85B (Rajagopalan et al., 2010), confirmed dnaA promoter is MtrA target (Li et al., 2010), and that the gene product encoded by lpq, which is located immediately downstream of mtrB, interacts with MtrB and possibly regulates its phosphorylation status (Nguyen et al., 2010).

Although the above studies suggest that the MtrAB system plays an important role in M. tuberculosis survival, several key questions remain with respect to the biochemical properties of the MtrAB system. For example, do MtrA and MtrB proteins interact and act as an intact signal transduction pair? Does MtrB exhibit autophosphorylation and phosphotransfer activities? What are the residues in MtrA that are critical for phosphorylation? Does a MtrA protein defective for phosphorylation interact with its targets proficiently? Finally, do MtrA merodiploid strains producing altered MtrA phosphorylation activity exhibit growth defects in the absence of phosphorylation activation signals? It is hoped that the answers to these questions as well as the identification of the conditions that cause MtrA to be constitutively active for phosphorylation could provide valuable insights into MtrA function.

2. Materials and methods

2.1 Strains and bacterial growth conditions

Escherichia coli strains were grown in Luria-Bertani (LB) broth or LB agar supplemented with ampicillin (Amp, 50 µg ml⁻¹), hygromycin (Hyg, 200 µg ml⁻¹) or ampicillin and 0.2% glucose. M. tuberculosis strains were propagated in Middlebrook 7H9 broth supplemented with OADC (oleic acid, albumin, dextrose, catalase and sodium chloride), 0.05% Tween 80 and hygromycin (Hyg at 50 µg ml⁻¹). Growth was monitored by measuring absorbance at 600 nm.

2.2 Mutagenesis and construction of mtrA merodiploid strains

For overproduction of MtrA in M. tuberculosis, we used the integration-proficient plasmid pJfr19 and placed the mtrA coding region downstream of the constitutively active acetamidase promoter as an NdeI-XbaI fragment, as previously described (Fol et al., 2006). Unless otherwise noted, PCR amplifications were carried with high fidelity deep vent DNA polymerase and genomic DNA as a template to replace the aspartic acid residues at the 13^th and 56^th positions. The primer combination MtrAD10A-F and MVM410-R was used to replace the aspartic acid at the 13^th codon with alanine to create plasmid pMZ3, whereas the primer combinations MVM409-F-MVM410-R and D53E(F)-D53(R) were used to replace the aspartic acid at the 56^th codon with glutamic acid, using an overlay PCR technique to create plasmid pDS3. Primers MtrAD10A-F and MVM410-R and template pMG129 (Fol et al., 2006) were used to replace the aspartic acids at codons 13 and 56 with alanine and asparagine, respectively, to create pMZ5, whereas the primer combination MVM-409-F-MVM-410R and the template pDS3 were used to replace the aspartic acids at codons 13^th and 56^th with alanine and glutamic acid, respectively, to create pDS6. All PCR products were sequenced to confirm sequence identity. The recombinant plasmids pMZ3, pMZ6, pMZ5, pDS3 and pDS6 were stably integrated at the attB locus of M. tuberculosis by electroporation to create the merodiploid strains Rv-MZ3, Rv-MZ5, Rv-DS3 and Rv-DS6, respectively. The sources of the M. tuberculosis mtrA merodiploid strain overproducing wild-type MtrA (Rv-78) and the phosphorylation-defective MtrA (Rv-129) were previously reported (Fol et al., 2006).

2.3 Purification of recombinant wild-type and mutant MtrA, MtrB and EnvZ proteins

For the overproduction and purification of recombinant wild-type and mutant MtrA proteins, respective mtrA coding regions were cloned into the pET-19b vector (Novagen) and transformed into the E. coli expression strain Arctic express (DE3) RIL (Stratagene). For protein overproduction, cultures were grown to an OD₆₀₀ of 0.6 and protein production was induced with 1 mM isopropyl-β-d-thiogalactopyranoside for 20 h at 10°C. MtrA proteins were purified under soluble conditions on Ni-NTA affinity columns (Qiagen) essentially following the manufacturer's recommendations. A truncated mtrB gene lacking the first 233 codons from the 5’ end was amplified using the primers MVM877-F and MVM878-R and then cloned as a fusion to gene for maltose binding protein. A recombinant plasmid expressing the MalE–EnvZ construct was a generous gift from Dr. M. Igo, University of California at Davis, CA. Soluble MtrB and EnvZ were purified as MalE–MtrB and MalE–EnvZ fusion proteins, respectively, from E. coli strain (DE3) RIL on amylose affinity columns following the manufacturer's instructions (NEB). MtrA, MtrB and EnvZ proteins were dialyzed for 4 h in storage buffer (20 mM sodium phosphate, 150 mM NaCl, 20% glycerol, 0.1 mM EDTA, and 1 mM DTT) and stored at -80°C.

2.4 Bacterial two-hybrid (BACTH) assay

Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) constructs were produced using the primers described in Table 1. The BACTH assay was carried out essentially as described by Karimova et al. (Karimova et al., 2005).. E. coli BTH101 recombinants with mtrA or mtrB plasmids or with control plasmids were selected on MacConkey agar supplemented with 100 mg/ml Amp and 50 µg/ml Km at 30°C for 24 to 36 h. For β-galactosidase activity measurements, cells grown in LB broth were permeabilized with 0.1% toluene and 0.01% sodium dodecyl sulfate, mixed with an equal volume of PM2 buffer (70 mM Na2HPO4, 30 mM NaH2PO4, 1 mM MgSO4 and 0.2 mM MnSO4, pH 7.0) and 100 mM b-mercaptoethanol. The reaction was started by the addition of 0.25 ml of 0.4% O-nitrophenol-β-galactoside (ONPG) in PM2 buffer, and the tube was incubated at 28°C for 5 min or until a visible yellow color developed. The reaction was stopped by the addition of 0.5 ml of 1 M Na₂CO₃, and the OD₄₂₀ was recorded. The enzymatic activity was defined as units per milliliter: 200 × [(OD₄₂₀ of the culture −OD₄₂₀ in the control tube)/minutes of incubation] × dilution factor. The specific activity of b-galactosidase is defined as units/mg dry weight bacteria, and 1 unit =1 nmol of ONPG hydrolyzed per min at 28°C. At least 5-fold higher b-galactosidase activity than that measured for BTH101 carrying a single gene and an empty vector was considered indicative of an interaction.

Table 1

PCR Primers

2.5 Autophosphorylation of MtrB and phosphotransfer to MtrA

MtrB autophosphorylation reactions (2 µM) were carried out in phosphorylation buffer (PB) containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl and 1 mM DTT supplemented with 20 mM MgCl₂ (Mg²⁺), 10 mM CaCl₂ (Ca²⁺) or both (Mg²⁺ and Ca²⁺). The reactions were initiated by adding ³²P-ATP and incubated for 2, 5, 10, 20 and 30 min at 37°C. The samples were removed, diluted with SDS-PAGE sample buffer and resolved in a 12% polyacrylamide gel under denaturing conditions, and protein bands were visualized using a BioRad Molecular Imager.

Phosphotransfer reactions using 2 µM MtrB were carried out in PB buffer supplemented with 20 mM MgCl₂ or 10 mM CaCl₂ plus 20 mM MgCl₂. MtrB autophosphorylation was initiated by adding ³²P-g-ATP; this was incubated at 37°C for 30 min and subsequently used in phosphotransfer reactions with 3 µM MtrA for 2 and 10 min. The samples were removed, diluted with SDS-PAGE sample buffer and resolved in a 12% polyacrylamide gel under denaturing conditions, and protein bands were visualized using a BioRad Molecular Imager.

2.6 Phosphorylation of MtrA by EnvZ Kinase

EnvZ kinase was phosphorylated for 5 min in the phosphorylation buffer described above. An aliquot of 2.5 µM phosphorylated EnvZ was mixed with 4 µM of either MtrA, MtrA_D13A, MtrA_D56E, MtrA_D10AD56E, MtrA_D13AD56N or MtrA_D56N, incubated for 15 min, resolved by SDS-PAGE, dried, exposed to a phosphoimager screen and then scanned in a Bio-Rad Molecular Imager.

2.7 Protein - DNA binding assay

EMSA experiments to detect MtrA binding to PfbpB were carried out using FITC-labeled promoter in buffer containing 50 mM Tris-HCl pH 7.0, 50 mM sodium chloride, 10 mM magnesium chloride, 10 mM calcium chloride, 1 mM DTT, 0.1 mM EDTA, 5% glycerol, 0.01% NP-40, 0.05 ng/µl sheared salmon sperm DNA, 250 nM poly dI/poly dC, 100 nM FITC-labeled PfbpB and 0.5 to 5 µM phosphorylated or non-phosphorylated MtrA as described (Rajagopalan et al., 2010). The samples were incubated for 15 min at 37°C and resolved by 5% TAE native gel electrophoresis (120 volts, 4C, 40 min). MtrA protein was phosphorylated by EnvZ in the presence of 20 mM cold ATP in PB for 15 min at 37°C. FITC-labeled PfbpB was prepared by amplification using the cloned PfbpB promoter and FAM-labeled T7 universal and Sp6 primers.

2.8 High performance gel filtration chromatography

MtrA proteins were separated on Superdex 200 10/300 GL (GE) with optimal resolution in the molecular weight range from 1.3 – 669 kDa. The column was equilibrated with buffer containing 50 mM Tris-HCl, 50 mM KCl, 20 mM MgCl2 and 1 mM DTT. Approximately 70 µg of MtrA protein was injected and resolved. BioRad gel filtration standards (Cat #151–1901) containing thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.3 kDa) were resolved, and their peak elution volumes plotted in a standard curve of V_e/V_o (elution volume/void volume) versus the log of molecular masses of proteins from gel filtration standards. Using this curve, the molecular size of MtrA protomer species was calculated.

2.9 RNA extraction and quantitative real-time PCR

Extraction of total RNA from broth-grown cultures of M. tuberculosis incubated for different periods of time was performed as described previously (Fol et al., 2006; Nair et al., 2009). DNA contamination was removed by treatment with DNase I (Ambion). cDNA was synthesized using 100 nM RT16S3 with RTAg85A2 primers and Superscript II reverse transcriptase (Invitrogen). Real-time PCR (Taqman chemistry) was performed in a Bio-Rad IQ5 Cycler using Taq DNA polymerase (NEB), the Taqman probes 16S-TP and 85B-TP (Biosearch Technologies), and reverse and forward primers (see Table 1). The calculated threshold cycle (Ct) value for each gene of interest was normalized to the Ct value for the 16S, and the fold expression was calculated using the formula: fold change = 2^Δ(ΔCt) (Fol et al., 2006; Nair et al., 2009). No reverse transcriptase (RT) reactions were included as negative controls. Expression data are the average from at least three independent RNA preparations, each reverse-transcribed and quantified by real-time PCR in triplicate. The real-time PCR conditions include initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing and extension at 60°C for 1 min.

3. Results and discussion

3.1 MtrB interactions with MtrA

A BACTH assay was used to evaluate interactions between the mtrA and mtrB gene products (Karimova et al., 2005). In these assays, the mtrA and mtrB genes were cloned as fusions to the T25 and T18 fragments of adenylate cyclase in two separate vectors and transformed into the reporter E. coli strain BTH101 (Karimova et al., 2005) Functional complementation of adenylate cyclase activity due to interactions between the partners leads to cAMP production and subsequent transcription of the lac reporter gene, which gives a distinct color to colonies growing on indicator agar plates. The strength of the interaction was also measured by assaying for b-galactosidase activity. As can be seen, transformants expressing mtrA from both vectors were blue, indicating a self-interaction similar to that observed when gcn4 is expressed from both vectors (Fig. 1A). Similarly, transformants expressing mtrA and mtrB from two different vectors were also blue, whereas those expressing mtrA plus a control vector or carrying two control vectors, pUT18C and pKNT25, were not (see Fig. 1). We also found that transformants expressing mtrB from both pUT18C and pKNT25 were not blue. Presumably, the self-interaction between MtrB protomers could be facilitated by their phosphorylation status, and signals that promote MtrB phosphorylation could be missing in the assay system used here. The extent of interaction between these proteins was evaluated by measuring the amount of b-galactosidase activity produced (Fig. 1B). Consistent with the observed interactions, constructs expressing mtrB-mtrA or mtrA-mtrA yielded b-galactosidase activity comparable to that of the positive control gcn4-gcn4. As expected, white transformant colonies did not show b-galactosidase activity (Fig. 1).

Fig. 1

BACTH assays showing MtrB and MtrA interactions. E. coli cells producing MtrA and MtrB proteins fused to the T18 and T25 subunits of adenylate cyclase were assessed for interaction strength by quantitating beta-galactosidase activity. The graph presents (more ...)

3.2 MtrB autophosphorylation and transphosphorylation activities

Histidine-aspartate sensor kinases exhibit autophosphorylation and transphosphorylation activities (Hoch, 2000). Our earlier studies showing that overexpression of mtrB along with mtrA reverses growth defects associated with mtrA overexpression in macrophages support the notion that MtrB kinase regulates the phosphorylation potential of MtrA RR (Fol et al., 2006). These studies assumed but have not proven that MtrA and MtrB work as an intact signal-transducing pair. To address this issue, we evaluated the autophosphorylation and transphosphorylation activities of MtrB kinase. MtrB is a 567-residue membrane-binding protein with two potential transmembrane domains (residues spanning 42–62 and 213–233), a HAMP linker (residues 215–284) and a His Kinase A phosphoacceptor (residues 302–519) domain (Aravind and Ponting, 1999; Bilwes et al., 1999). Hence, to evaluate phosphorylation activity, we amplified a fragment encoding a soluble derivative of the protein spanning residues 234 to the end, cloned it as a fusion to Maltose Binding Protein, purified the recombinant protein on amylose resin and characterized it. Initial phosphorylation experiments were performed in buffers containing the divalent cations Mg²⁺, Ca²⁺ or Mg²⁺ plus Ca²⁺ (Fig. 2A). A time-dependent increase in MtrB phosphorylation was noted with all metal ions (Fig. 2, panels i and ii). Densitometric analysis revealed that buffers containing Mg²⁺ plus Ca²⁺ showed higher levels of autophosphorylation activity compared to buffer containing Mg²⁺ alone (Fig. 2A-ii). We noted that at longer incubation times, i.e., 10 min and beyond, MtrB protein aggregated in buffers containing Ca²⁺ (data not shown).

Fig. 2

Autophosphorylation and phosphotransfer properties of MtrB in the presence of magnesium and calcium. A. Enhanced autophosphorylation of MtrB in the presence calcium and magnesium. i) Phosphorylation reactions were carried for 2, 5, 10, 20 or 30 minutes (more ...)

Next, we examined the phosphotransfer activity of MtrB. In these experiments, MtrB was first phosphorylated by incubating in the presence of ³²P-ATP for 30 min followed by the addition of MtrA. Controls received buffer in place of MtrA. Samples were removed after 2 and 10 min of contact with MtrB. As a positive control, EnvZ protein was phosphorylated for 5 min and then incubated with MtrA. Consistent with the earlier published data (Fol et al., 2006), incubation of radio-labeled ATP with EnvZ led to MtrA phosphorylation (Fig. 2B, lane marked MtrA+EnvZ). Similar to EnvZ, MtrB kinase also promoted phosphorylation of MtrA in buffer containing Mg⁺², but not in buffer containing both Mg²⁺ and Ca²⁺. These results indicate that although the divalent metal ion Ca²⁺ promotes MtrB phosphorylation, it interferes with MtrB transphosphorylation activity. This intriguing result signals a possibility that the phosphorylation potential of MtrB kinase could be regulated by MtrB interactions with divalent Ca²⁺ ions. This could in turn dictate the level and extent of MtrA phosphorylation and possibly the signal output. This finding may have important implications for understanding Mtb pathogen survival in vivo. For example, it is believed that Mtb survival upon infection is critically dependent on the metal ion composition of the intraphagosomal environment (Wagner et al., 2005a; Wagner et al., 2005b). Reports indicate that the metal ion composition of vacuoles containing pathogenic and nonpathogenic mycobacterial species differs (Wagner et al., 2005a) It is unknown whether divalent ions other than Ca²⁺ interfere with MtrB transphosphorylation activity. Presumably, the ratio of Ca²⁺ to other metal ions influences the signal output of the MtrAB system during intracellular growth. Alternatively, in the presence of excess Ca²⁺, MtrB~P could phosphorylate other RR proteins and/or perform functions independently of MtrA. These possibilities are not mutually exclusive. Further studies are required to address these issues.

3.3 Amino acid residues important for MtrA phosphorylation

Mutagenesis studies with several bacterial RRs have shown that replacement of the conserved D residues with select amino acids modulates RR phosphorylation potential and associated phenotypes (reviewed in (Hoch, 2000; Stock et al., 2000)). Such replacements in some cases could lead to constitutive activation of the RR. Constructing and characterizing MtrA proteins with altered phosphorylation properties that potentially influence MtrA activity, and hence M. tuberculosis survival, could provide valuable insights into the MtrAB signal transduction system. Recent MtrA crystal data indicate that D13 and D56 are conserved residues in the receiver domain (Friedland et al., 2007). To begin addressing this issue, we initially focused on the D residues at the 13^th and 56^th positions, overproduced mutant proteins, purified them as His-fusion proteins and determined their phosphorylation activity. Unlike wild-type MtrA, all the mutant proteins failed to be phosphorylated by the EnvZ kinase (Fig. 3). These results confirm that the D residues at positions 13 and 56 are important for MtrA phosphorylation (Fig. 3). Presumably, replacement of these aspartic acid residues with alanine, glutamine or asparagine inactivates MtrA protein for phosphorylation or keeps it in a state that no longer accepts the phosphate group. The latter possibility implicates that the mutant proteins behave differently compared to the wild-type protein with respect to their ability to bind targets and possibly modulate target gene transcription.

Fig. 3

In vitro phosphorylation activity of various MtrA proteins. Recombinant mutant MtrA proteins were purified, and approximately 3 µM of each protein was evaluated for its ability to exhibit phosphorylation in the presence of 2 µM EnvZ and (more ...)

3.4 Interaction of MtrA proteins with the fbpB promoter

One consequence of RR phosphorylation is activation of its output domain that promotes RR binding to DNA and modulates target gene expression (Hoch, 2000). We recently demonstrated that promoters for the secreted antigen 85B (PfbpB) and dnaA (PdnaA) and the origin of replication (oriC) are bona fide MtrA targets and that phosphorylated MtrA binds preferentially to PfbpB and oriC (Rajagopalan et al., 2010). Accordingly, we evaluated MtrA binding to PfbpB by EMSA. Preliminary experiments revealed that MtrA_D13AD56N and MtrA_D13AD56E proteins aggregated during incubation with DNA, and thus, the DNA-protein complexes remained in the wells (not shown). Consequently, EMSA experiments were carried out with MtrA_D56E, MtrA_D13A and MtrA_D56N (Fig. 4). As expected, MtrA and its phosphorylated derivative MtrA~P (produced by incubation with EnvZ and ATP) bound to PfbpB and retarded its mobility, although MtrA~P resulted in greater retardation at all concentrations tested (Fig. 4). MtrA_D13A bound PfbpB both in the presence and absence of EnvZ and ATP; however, binding appeared to be favored in the absence of ATP and EnvZ. Binding under these conditions was as proficient as that with wild-type MtrA in the presence of ATP and EnvZ (Fig. 4).

Fig. 4

EMSA showing interactions between MtrA and PfbpB. Approximately 100 femtomoles of FITC-labeled PfbpB DNA was incubated with increasing amounts of MtrA protein mixed with or without EnvZ and ATP was incubated for 15 min in the presence of buffer containing (more ...)

MtrA_D56E showed significant binding, but only in the absence of ATP and EnvZ (Fig. 4). Also, two shifted bands were detected at some protein concentrations (see Fig, 4, lanes marked 2 and 3 corresponding to no ATP and EnvZ). On the other hand, phosphorylation-defective MtrA_D56N did not bind PfbpB (Fig. 4). Presumably, the ability of MtrA to undergo phosphorylation could be critical for its interaction with the target and that replacement of the conserved aspartic acid residue with asparagine made the protein inert for interaction with its target. On the other hand the analogous D56E mutation at the phosphorylation site in other RR that have been investigated, i.e. NtrC, OmpR and CtrA, mimics aspartate-phosphoryl state, activates RR and bypasses the requirements for the cognate sensor kinase (Domian et al., 1997; Klose et al., 1993; Lan and Igo, 1998). Phosphorylation of RR often enhances its DNA binding affinities and promotes cooperative binding at two adjacent recognition sequences. The fpbB promoter region has been predicted to have four direct MtrA repeat binding motifs (Rajagopalan et al., 2010). Thus, the appearance of two distinct shifted bands in reactions incubated in the absence of EnvZ supports the notion that D53E mutation affected protein confirmation in the signal receiving domain of RR, possibly mimics phosphorylation state, hence promotes cooperative DNA-binding. The observed differences in the PfbpBDNA-binding between MtrA_D13A and MtrA_D56E could reflect differences in the activities of the protein preparations.

The observed differences in the binding of MtrA proteins to PfbpB (notably in the absence of ATP and EnvZ) could reflect changes in protein conformation. Such changes could involve protein dimerization and higher order oligomerization upon phosphorylation. This is not entirely unexpected because RRs, specifically OmpR/PhoP family proteins, often exhibit a propensity to dimerize upon phosphorylation (Bilwes et al., 1999; Hoch, 2000), and MtrA belongs to the OmpR/PhoP family of RRs. Hence, to address the consequences of mutations on protein conformation, we compared the elution profiles of the different MtrA proteins by size exclusion chromatography on a Superdex S200 column in buffers lacking ATP and EnvZ kinase. A protein standard mix containing tyroglobulin (670 kDa), g-globulin (158 kDa), ovalbumin (44 kDa) and myoglobulin (17 kDa) resolved into distinct peaks (Fig. 5, panel A). Wild-type and mutant MtrA proteins resolved into single peaks, and the positions of the peaks corresponded to monomers (Fig. 5, panels B–E). These results indicate that replacement of the D residues at 13 and 56 positions with select amino acids does not significantly affect protein conformation. MtrA incubated with ATP and EnvZ also failed to show a significant change in peak position, indicating the absence of visible dimers and oligomers, although MtrA~P produced under these conditions showed enhanced binding to its target (Fig. 4). Presumably, the extent of MtrA phosphorylation provided by EnvZ is not sufficient to promote either dimerization or higher order oligomerization. Notably, the recent MtrA crystal studies suggest that the orientation of the regulatory and DNA-binding domains is such that it stabilizes the inactive conformation of MtrA and reduces the rate of phosphorylation and thus regulates the MtrA activity (Friedland et al., 2007).

Fig. 5

Size exclusion chromatography of recombinant MtrA proteins on Superdex 200 10/300 GL. A. Top graph: Elution profiles of the molecular mass standards. Bottom graph: Peak elution volumes of protein standards [thyroglobulin (670 kDa), γ-globulin (more ...)

3.5 M. tuberculosis mtrA merodiploid strains show growth defects in nutrient broth

Above, we showed that although MtrA_D13A, MtrA_D56E and MtrA_D56N proteins are each defective for phosphorylation, their interactions with their PAg85B target differ. MtrA RRs can have multiple targets, and it is likely that the mutant MtrA proteins both interact with and modulate the expression of these targets differently, which could in turn affect the growth and proliferation of the respective strains. Because mtrA is an essential gene, to address this possibility, we created merodiploid strains and evaluated their growth patterns in nutrient broth (Fig. 6). Consistent with our previously published data, wild-type M. tuberculosis (Rv-19) and M. tuberculosis overproducing phosphorylation-proficient (Rv-78) and phosphorylation-defective (Rv-129) MtrA merodiploids grew similarly in nutrient broth (Fig. 6A) (Fol et al., 2006). In contrast, Rv-MZ3 and Rv-DS3 merodiploids producing MtrA_D13A and MtrA_D56E grew normally during early periods but showed a growth defect at later periods and reached stationary growth early.

Fig. 6

Phenotypes of mtrA merodiploid strains. (A). The growth of M. tuberculosis merodiploid strains was monitored by measuring absorbance at 600 nm. Representative data from three experiments are shown. (B) Quantitative real-time PCR analysis of Ag85B gene (more ...)

3.6 The expression of fbpB is downregulated in Rv-DS3

Earlier studies with Rv-78 merodiploids indicated that signals promoting MtrA phosphorylation are enriched during intramacrophage growth (Fol et al., 2006) and that fbpB expression is sharply downregulated in Rv-78 upon infection (Rajagopalan et al., 2010). It is likely that the mutant MtrA proteins defective for phosphorylation modulate the expression of target genes independent of phosphorylation signals. Accordingly, we hypothesized that the expression of MtrA target genes (including fbpB) could be affected in Rv-MZ3 and Rv-DS3 merodiploids relative to wild-type and Rv-78. To gain insight into this issue, we characterized one of the merodiploids (Rv-MZ3) further as a proof of principle, and we evaluated fbpB expression relative to 16S rRNA at 3 and 8 days of growth in both Rv-19 and Rv-78 (Fig. 6B). As shown in Fig. 6B, fbpB expression was downregulated 4.5- and 16.8 -fold relative to wild type at days 3 and 8, respectively, whereas it was modestly lower in Rv-78 by 1.8- and 2.5-fold at days 3 and 8, respectively, relative to wild-type.

We conclude from the above results that certain mutations in the signal-receiving domain of MtrA that impair its phosphorylation potential interfere with growth and possibly exhibit a dominant-negative phenotype. Although more detailed studies with other mutations are required, our results showing growth retardation and downregulation of fbpB expression in broth – grown Rv-MZ3 tend to indicate that the observed phenotypes are reminiscent of Rv-78 in macrophages. Because MtrA_D13A is defective for phosphorylation and binds to PfbpB similarly with or without EnvZ and ATP (Fig. 4), we propose that MtrA_D13A acts as a constitutively active protein and exerts its effects in the absence of signals that activate the MtrAB system. MtrA could have many targets, and in the current study, only its interaction with the PfbpB target was evaluated. It is likely that the interactions of MtrA_D13A with other target promoters are also modulated in vivo, thereby influencing growth. Further studies are required to evaluate this possibility. Nonetheless, our studies identified a collection of useful mutants, and their detailed characterization in vitro and in vivo could improve our understanding of the MtrAB signal transduction process.

Acknowledgments

The work was supported in part by NIAID grants RO1AI48417 (MR) and RO1AI73596, AI84734 (MM). We thank the members of Madiraju and Rajagopalan labs for helpful suggestions and stimulating discussions.

Footnotes

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References

Aravind L, Ponting CP. The cytoplasmic helical linker domain of receptor histidine kinase and methyl-accepting proteins is common to many prokaryotic signalling proteins. FEMS Microbiol Lett. 1999;176:111–116. [PubMed]
Bilwes AM, et al. Structure of CheA, a signal-transducing histidine kinase. Cell. 1999;96:131–141. [PubMed]
Cangelosi GA, et al. The two-component regulatory system mtrAB is required for morphotypic multidrug resistance in Mycobacterium avium. Antimicrob Agents Chemother. 2006;50:461–468. [PMC free article] [PubMed]
Cole ST, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998;393:537–544. [PubMed]
Domian IJ, et al. Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle. Cell. 1997;90:415–424. [PubMed]
Fol M, et al. Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulator. Mol Microbiol. 2006;60:643–657. [PubMed]
Friedland N, et al. Domain orientation in the inactive response regulator Mycobacterium tuberculosis MtrA provides a barrier to activation. Biochemistry. 2007;46:6733–6743. [PMC free article] [PubMed]
Hoch JA. Two component and phosphorely signal transduction. Curr. opin. Microbiol. 2000;3:165–170. [PubMed]
Karimova G, et al. Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J Bacteriol. 2005;187:2233–2243. [PMC free article] [PubMed]
Klose KE, et al. Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein. J Mol Biol. 1993;232:67–78. [PubMed]
Lan CY, Igo MM. Differential expression of the OmpF and OmpC porin proteins in Escherichia coli K-12 depends upon the level of active OmpR. J Bacteriol. 1998;180:171–174. [PMC free article] [PubMed]
Li Y, et al. The characterization of conserved binding motifs and potential target genes for M. tuberculosis MtrAB reveals a link between the two-component system and the drug resistance of M. smegmatis. BMC Microbiol. 2010;10:242. [PMC free article] [PubMed]
Moker N, et al. Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection. Mol Microbiol. 2004;54:420–438. [PubMed]
Moker N, et al. In vitro analysis of the two-component system MtrB-MtrA from Corynebacterium glutamicum. J Bacteriol. 2007 [PMC free article] [PubMed]
Nair N, et al. Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants. Mol Microbiol. 2009;71:291–304. [PMC free article] [PubMed]
Nguyen HT, et al. A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug resistance, cytokinetic control and cell wall homeostasis in Mycobacterium. Mol Microbiol. 2010;76:348–364. [PubMed]
Rajagopalan M, et al. Mycobacterium tuberculosis origin of replication and the promoter for immunodominant secreted antigen 85B are the targets of MtrA, the essential response regulator. J Biol Chem. 2010;285:15816–15827. [PubMed]
Stock AM, et al. Two-component signal transduction. Annu Rev Biochem. 2000;69:183–215. [PubMed]
Via LE, et al. Elements of signal transduction in Mycobacterium tuberculosis: in vitro phosphorylation and in vivo expression of the response regulator MtrA. J Bacteriol. 1996;178:3314–3321. [PMC free article] [PubMed]
Wagner D, et al. Elemental Analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-Containing Phagosomes Indicates Pathogen-Induced Microenvironments within the Host Cell’s Endosomal System. J Immunol. 2005a;174:1491–1500. [PubMed]
Wagner D, et al. Changes of the phagosomal elemental concentrations by Mycobacterium tuberculosis Mramp. Microbiology. 2005b;151:323–332. [PubMed]
Zahrt TC, Deretic V. An essential two-component signal transduction system in Mycobacterium tuberculosis. J Bacteriol. 2000;182:3832–3838. [PMC free article] [PubMed]