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 Nde
I 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 13th
positions. The primer combination MtrAD10A-F and MVM410-R was used to replace the aspartic acid at the 13th
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 56th
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 13th
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 att
B 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 mtr
A 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 . The BACTH assay was carried out essentially as described by Karimova et al. (Karimova et al., 2005
).. E. coli
BTH101 recombinants with mtr
A or mtr
B 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 Na2
, 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.
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, MtrAD13A, MtrAD56E, MtrAD10AD56E, MtrAD13AD56N or MtrAD56N, 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 Pfbp
B 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 Pfbp
B 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 Pfbp
B was prepared by amplification using the cloned Pfbp
B 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 Ve/Vo (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 ). 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 = 2Δ(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.