Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis
© Sasindran et al; licensee BioMed Central Ltd. 2011
Received: 18 August 2010
Accepted: 25 February 2011
Published: 25 February 2011
Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they participate in spore formation, stress adaptation, ribosome assembly and chromosomal partitioning. This study was undertaken to investigate the biochemical and physiological characteristics of Obg in Mycobacterium tuberculosis, which causes tuberculosis in humans.
We overexpressed M. tuberculosis Obg in Escherichia coli and then purified the protein. This protein binds to, hydrolyzes and is phosphorylated with GTP. An anti-Obg antiserum, raised against the purified Obg, detects a 55 kDa protein in immunoblots of M. tuberculosis extracts. Immunoblotting also discloses that cultured M. tuberculosis cells contain increased amounts of Obg in the late log phase and in the stationary phase. Obg is also associated with ribosomes in M. tuberculosis, and it is distributed to all three ribosomal fractions (30 S, 50 S and 70 S). Finally, yeast two-hybrid analysis reveals that Obg interacts with the stress protein UsfX, indicating that M. tuberculosis Obg, like other bacterial Obgs, is a stress related protein.
Although its GTP-hydrolyzing and phosphorylating activities resemble those of other bacterial Obg homologues, M. tuberculosis Obg differs from them in these respects: (a) preferential association with the bacterial membrane; (b) association with all three ribosomal subunits, and (c) binding to the stress protein UsfX, rather than to RelA. Generation of mutant alleles of Obg of M. tuberculosis, and their characterization in vivo, may provide additional insights regarding its role in this important human pathogen.
GTP-binding proteins are found in all living organisms, and they play critical roles in fundamental processes such as cell proliferation, development, signal transduction and protein translation [1, 2]. In general, these proteins are hydrolase enzymes that convert GTP into GDP, allowing transfer of the GTP terminal phosphate group to a target protein. As a consequence of this transfer, the highly conserved domains (G1, G2, G3, G4 and G5) of GTP-binding proteins undergo conformational changes that are detected by downstream effector proteins [3, 4], leading to specific outcomes.
Comparison of bacterial genomes, across all taxa, has shown that at least eleven highly conserved GTP-binding proteins are present in prokaryotes . Among these, the Obg/GTP1 subfamily of monomeric GTP binding proteins is of special significance, because these proteins exist not only in prokaryotes but also in eukaryotes . The gene encoding Obg was first identified in Bacillus subtilis . Obg orthologues were subsequently discovered in Streptomyces griseus , Streptomyces coelicolor , Caulobacter crescentus , Echerichia coli  and Vibrio harveyi . While orthologues of Obg in C. crescentus and V. harveyi are known as CgtA, the orthologue of Obg in E. coli is called ObgE. Bacterial Obg display intrinsic GTPase activity and autophosphorylate with GTP, as does the eukaryotic signaling molecule Ras, which is a GTP-binding protein. Because of this, Obg has been considered to be a potential bacterial signaling molecule [8, 13].
Several published studies have attributed diverse functions to Obg in different bacterial species. In B. subtilis, for example, Obg is necessary for the transition from vegetative growth to stage 0 or stage II of sporulation . Sporulation is a complex process in this species and is controlled by multiple components including phosphorelay. It appears that Obg is one of the components that modulate the sporulation-related phosphorelay by an undefined mechanism . In addition to its activity in B. subtilis, Obg plays critical roles in developmental events in other bacteria, e.g. aerial mycelium formation and sporulation in Streptomyces griseus  and S. coelicolor . In these two species, sporulation has a tight relationship with changes in the intracellular GTP-to-GDP ratio, and bacterial Obgs are considered to be stress sensors for intracellular GTP-GDP changes reflecting energy balance in the cells. It has been proposed that high levels of Obg-GTP maintain vegetative division of sporulating bacteria and prevent sporulation, while high levels of Obg-GDP promote sporulation .
Obg is required for the activation of B. subtilis SigB in response to physical stress. This activation occurs via Obg's physical interaction with upstream Rsb regulators of SigB . Further, the GTP-binding pocket of crystallized Obg of B. subtilis contains guanosine 5' diphosphate, 3' phosphate (ppGpp) . ppGpp is a guanosine nucleotide known as an alarmone in bacteria. Alarmones are produced in response to amino acid starvation, and they act as signaling intermediates to slow cell growth or to initiate stress-induced differentiation pathways, including sporulation. In bacteria, the synthesis of ppGpp is performed by two enzymes, called RelA and SpoT [17–19]. In E. coli, SpoT is one of the proteins known to interact with Obg . In V. cholerae, depletion of the Obg homologue CgtA results in a global gene expression pattern reflecting the low-nutrient stress reaction called the "stringent" response . In V. cholerae, CgtA interacts with SpoT, and this interaction decreases SpoT activity leading to the repression of the stringent response . Another interesting example of Obg's association with stress comes from the pathogen Legionella pneumophila, where its expression is elevated during intracellular survival .
Recent studies indicate that Obg associates with ribosomes of bacteria and interacts with ribosomal proteins. In B. subtilis, Obg coelutes with ribosomal proteins and interacts specifically with the ribosomal protein L13, a component of the 50 S ribosomal subunit . The Obg orthologues of C. crescentus , V. harveyi  and E. coli [20, 26] also cofractionate with the 50 S ribosomal subunit. Finally, bacterial Obg has also been implicated in chromosomal partitioning  and replication regulation .
Mycobacterium tuberculosis is an intracellular pathogen and causative agent of tuberculosis in humans. The recent emergence of multidrug (MDR-TB) and extremely drug resistant (XTR-TB) M. tuberculosis strains now poses serious threats to people in the developing world , and combating the disease requires the development of new anti-tuberculosis drugs. However, design and development of new drugs for TB largely depends upon the identification and characterization of novel drug targets in M. tuberculosis. The fact that Obg is an essential protein for growth in bacteria, including M. tuberculosis , and its association with ribosomes makes it a potential target for future antimicrobials [29, 30]. Thus, this study was undertaken to understand the basic properties of Obg of M. tuberculosis.
Results and Discussion
Overexpressed M. tuberculosisObg binds to, and hydrolyzes, GTP
To verify whether the overexpressed Obg of M. tuberculosis can interact with GTP, we performed GTP-UV-crosslinking experiments . The autoradiogram in Figure 1B shows that His10-Obg binds physically to [α32P]-GTP. Exposure of the reaction mixtures to UV irradiation for 0, 30 and 60 min revealed that binding of GTP with His10-Obg is increased between 0 and 30 min of exposure, but not after 30 min (Figure 1B). When the reactions were performed in the presence of unlabeled GTP (5 mM), crosslinking of His10-Obg to GTP is inhibited, while addition of large amounts of unlabeled ATP (500 mM) have little effect on His10-Obg binding with labeled GTP (Figure 1B). This observation adds to existing evidence that M. tuberculosis Obg has an inherent specificity for guanine nucleotides, as do the Obg orthologues in C. crescentus , B. subtilis  and S. griseus .
To determine whether the overexpressed Obg can hydrolyze GTP, we incubated His10 -Obg with radiolabeled GTP ([γ-32P] GTP), and measured the release of phosphate (32Pi) after 3 hours. Figure 1C shows that His10-Obg readily hydrolyzes GTP, and that this hydrolysis is inhibited by the addition of unlabeled GTP (5 mM), indicating that unlabeled GTP competes with labeled GTP for the enzyme. Addition of unlabeled ATP (5 mM) has no effect on the hydrolysis of labeled GTP (Figure 1C), indicating that Obg hydrolyzes specifically GTP. The effect of cold GTP in inhibiting the hydrolysis of radiolabeled GTP was not as pronounced as its effect in inhibition of GTP crosslinking (Compare Figure 1B and Figure 1C). This is most likely due to the differences in the positions of the radiolabeled phosphates used in these two reactions. While the reaction mixture in the crosslinking experiment (Figure 1B) had 10 μCi (0.033 μM) of [α-32P] GTP, the reaction mixture in the hydrolysis experiment had 25 μCi (0.040 μM) of [γ-32P] GTP. In addition, the incubation times for these two experiments were different (1 h for GTP crosslinking vs. 3 h for GTP hydrolysis).
Autophosphorylation of His10-Obg
Expression of M. tuberculosisObg is growth-dependent, and Obg is associated with the membrane fraction
Notably, Obg expression does change in cultures of M. tuberculosis over the course of cell growth. Obg expression is markedly increased from early log phase to the stationary phase, with a drop in expression at late stationary phase (Figure 3B). Comparison of the Obg band densities discloses that expression of Obg at later growth phases (1.645 OD600 nm ) is approximately five fold higher than it is at earlier phases (0.220 OD600 nm), even before the drop in expression at late stationary phase. Together these results indicate that the expression of Obg in M. tuberculosis is growth-regulated, being increased as the cells begin rapid division in the log phase, and maintained at high levels until late in the stationary phase. However, whether increased levels of Obg with increased growth of M. tuberculosis is due to increased expression of Obg, or to accumulation of Obg, remains to be determined. Obg expression in E. coli is also high in log phase growth, but decreased in the stationary phase .
In S. griseus  and E. coli , Obg and its orthologues are found in both the cytoplasmic and membrane fractions. In B. subtilis, however, Obg is mainly associated with the cytoplasm . To determine where Obg resides in M. tuberculosis, we isolated soluble and membrane fractions from whole bacteria, and subjected them to immunoblot analysis. Figure 3C shows that Obg is associated mostly (over 90%) with the membrane fraction, although detectable amounts are also present in the soluble fraction. In contrast, SigH of M. tuberculosis, which was used as a control here, exhibits almost equal distribution between these two fractions. It has been reported that membrane fraction-bound Obg in S. coeliocolor  and in E. coli  is lost from this fraction if the extraction buffer contains 5 mM EDTA. The buffer we use for M. tuberculosis membrane preparations has 10 mM EDTA, however, and Obg is associated with this fraction whether or not EDTA is present (not shown). The EDTA-resistant association of M. tuberculosis Obg to the membrane fraction may reflect a function associated with signaling, and involving divalent cations. Interestingly, Obg is absent from detergent-extracted M. tuberculosis membrane  and cell wall  proteins, suggesting that Obg's association with the membrane may be due to its interaction with other membrane protein(s).
M. tuberculosisObg associates with ribosomal fractions
M. tuberculosisObg interacts with UsfX
Interaction of Obg with stress related proteins in the yeast two-hybrid system.
SD Minimal Medium
Mel-l (α-gal) in
Mel-1 (α-gal) in
3.512 ± 0.709
2.367 ± 0.354
2.172 ± 0.448
In B. subtilis, the activation of SigB in response to stress depends upon its association with, and dissociation from, of RsbW. In turn, this is governed by the phosphorylation state of RsbW . The UsfX protein of M. tuberculosis is believed to have similar interaction with its cognate sigma factor SigF . Whether the interaction of Obg with UsfX affects the phosphorylation state of UsfX is unknown. Additional studies assessing the interaction of Obg and UsfX in vitro, and careful examination of phosphate exchange in vivo, may throw light on this part of Obg function. The Obg/CgtA proteins of E. coli and V. harveyi interact with SpoT, a stringent response regulator and a relative of RelA, which responds to starvation. The fact that Obg of M. tuberculosis fails to interact with RelA suggests that the stress response roles of Obg of M. tuberculosis differ from those of its homologues in other bacteria.
Overexpression of Obg affects late log phase growth of M. tuberculosis
Our data reveal that M. tuberculosis Obg has characteristics that are common to its homologues in other bacteria, in addition to properties that are unique. Generation and characterization of mutant alleles of M. tuberculosis Obg should provide additional insights to the role of Obg in this important human pathogen, and toward identification of antimicrobials that reduce its ability to promote M. tuberculosis survival.
Bacteria and yeast strains and their growth conditions
M. tuberculosis H37Rv was grown either in Middlebrook 7H9 broth medium containing Tween (0.05%) and OADC (10%) (7H9-TW-OADC) broth, or in Middlebrook 7H10 agar medium containing Tween (0.05%) and OADC (10%) (7H10-TW-OADC). M. tuberculosis strains harboring plasmids were grown in the above media containing the antibiotic kanamycin (25 μg/ml) or hygromycin (50 μg/ml). E. coli strains containing plasmids were grown in LB broth or LB agar plates with the antibiotic(s) ampicillin (100 μg/ml), kanamycin (25 μg/ml) or both. Unless specified, all bacteria were grown at 37°C. The yeast strain AH109 was grown at 30°C in YPD broth or in agar supplemented with adenine hemisulphate (0.003%).
List of primers used in this study.
List of plasmids used in this study.
oriColE1, lacZα, Plac, aph, AmpR
oriE, oriM, Phsp60, aph
Stover et al, 1991
pMV261-Rv2440c full orf
oriE, lacI, PT7, AmpR
pET16B-Rv2440c full orf
oriColE1, ori2 μ, LEU1, PADH1::GAL4' activator domain::MCS AmpR
oriColE1, ori2 μ, TRP1, PADH1::GAL4' binding domain::MCS KmR
SV40 large T-antigen(84-708) in pGADT7
Murine p53(72-390) in pGBKT7
Human lamin C(66-230) in pGBKT7
pGADT7-Rv2853c full orf
pGBKT7-Rv3286c full orf
Parida et al, 2005
pGADT7-Rv3287c full orf
Parida et al, 2005
pGBKT7-Rv2440c full orf
Overexpression of M. tuberculosis Obg in E. coliand production of antiserum
The E. coli-overexpressed Obg protein of M. tuberculosis was purified in its native condition. The plasmid construct pTBOBGE was transformed into E. coli strain BL21(DE3). A single transformant colony was selected and grown in 2 ml of LB broth overnight. One ml of this overnight culture was inoculated into 250 ml LB broth and grown to log phase (0.350 OD at 590 nm) at 37°C. IPTG (1 mM) was then added to the culture to induce overexpression of Obg, and the culture was grown for an additional 3 h. Afterwards, E. coli cells were harvested by centrifugation (5,000 g for 10 min at 4°C) and stored overnight at -80°C. The pellet was resuspended in 5 ml of lysis buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM Imidazole) containing 1 mg/ml of lysozyme, incubated on ice for 30 min and the cells disrupted by sonication. The lysate was centrifuged at 12,000 g, and the supernatant was loaded on to a 2 ml Ni-NTA column (Qiagen). After washing the column with 50 ml of wash buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 20 mM Imidazole), the column- bound Obg protein (His10-Obg) was eluted with 2 ml of elution buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 250 mM Imidazole). The eluted fraction was dialyzed against 2 L of 20 mM Tris-HCl pH 8.0 containing 5% glycerol. About 100 μg of the resulting purified Obg was used to immunize a single rabbit to generate polyclonal antiserum, according to standard protocols. The rabbit received two booster doses of similar amounts of protein at two week intervals before collecting the serum two weeks after the last booster dose.
Crosslinking of the Obg protein with GTP was done by mixing Ni-NTA-purified M. tuberculosis His-tagged Obg (His10-Obg) (5 μg) with a 40 μl cross-linking mixture (20 μCi of [α32P]-dGTP, 1 mM ATP, 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2 and 1% Triton X-100). Eppendorf tubes containing the mixture were kept for 1 h at 4°C in a dark chamber, and then placed on ice over a Petri dish to expose them to UV light (256 nm) for different time periods. Crosslinking of Obg with GTP was assessed after separating the crosslinked complexes on SDS-PAGE, transferring the proteins from the gel onto nitrocellulose membranes, and exposure of the membranes to X-ray film to detect the presence of 32P in the protein bands.
GTPase activity of Obg
To determine whether M. tuberculosis can hydrolyze GTP, we added [γ-32P]GTP to purified His10-Obg, following the method of Welsh et al . The reactions were conducted in 100 μl volumes containing 50 mM Tris pH 8.5, 0.1 mM EDTA, 1.5 mM MgCl2, 200 mM KCl, 10% glycerol, 25. μCi of [γ-32P]GTP and 7 μg of His10-Obg. These reactions were incubated at 37°C for 3 h, and then terminated by the addition of 700. μ1 of ice cold 20.mM phosphoric acid (pH2. 0) containing 5% activated charcoal. The charcoal was sedimented by centrifugation, and 100 μl of the remaining supernatant used to measure the 32Pi released. GTPase activity was expressed as 32Pi released (cpm)/μg protein/hour.
To determine whether M. tuberculosis Obg is autophosphorylated in the presence of GTP, His10-Obg (5 μg) was incubated with 10. μCi of [γ-32P]GTP in a 25 μl reaction mixture containing 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1.5 mM MgCl2, 100 mM KCl and 10% glycerol at 37°C. The reactions were arrested at different time points by the addition of SDS-PAGE sample buffer. The samples from different time points were subjected to SDS-PAGE and transferred to nitrocellulose membranes, and autophosphorylation of the Obg protein was visualized by autoradiography.
Soluble and membrane fractions
Soluble and membrane fractions of M. tuberculosis were prepared as described . Briefly, M. tuberculosis cells were grown to 0.6-1.0 OD (at 600 nm) in 400 ml of 7H9-OADC-TW broth. The cells were then harvested by centrifugation at 5,000 g. The pellet was resuspended in 25 ml of 20 mM sodium phosphate-10 mM EDTA (pH 7.0) buffer, and spun again at 5,000 g to remove the medium completely. The pellet was then suspended in 4 ml of 20 mM sodium phosphate-10 mM EDTA buffer containing a protease inhibitor cocktail (Sigma), and divided into four 2 ml screw cap tubes with O-rings containing silica beads. The tubes were cooled on ice, and then the cells were disrupted using a bead beater for two 1-min cycles, with a 30 second interval between them. The tubes were chilled on ice for 5 min and then centrifuged at 12,000 g at 4°C for 15 min. The resulting supernatants were pooled, transferred to 4 ml centrifuge tubes and spun at 49,000 g for 4 h at 4°C. These supernatants (soluble fraction) were transferred to fresh tubes for analysis, while the pellet (membrane fraction) was washed once with 4 ml of 20 mM sodium phosphate-10 mM EDTA buffer and resuspended in 0.5 ml of the same buffer. Protein concentrations in both the soluble and membrane fractions, and in the unseparated lysates, were determined by the BCA method (Pierce) before subjecting them to electrophoresis.
Preparation of ribosomal fractions
M. tuberculosis H37Rv cells were grown in 100 ml of 7H9-TW-OADC broth at 37°C. When the OD of the cultures reached to 0. 6 -1.0 (at 600 nm), the cells were harvested by centrifugation, resuspended in 2 ml of buffer A (10 mM Tris-HCl, pH 7.6, 10 mM magnesium acetate, 100 mM ammonium acetate, 6 mM β-mercaptoethanol, and 2 mM PMSF), and disrupted by bead beating as described earlier. The lysate was then centrifuged at 12,000 g for 15 min. The clear supernatant was collected and its protein concentration determined. About 500 μg of this protein was loaded onto a 10-40% sucrose gradient (total volume 4 ml) made in buffer B (10 mM Tris-HCl, pH 7.6, 1 mM magnesium acetate, 100 mM ammonium acetate, 6 mM β-mercaptoethanol, and 2 mM PMSF). The gradient was centrifuged at 90,000 g for 20 h. The gradients were then aliquoted into 250 μl fractions, and the absorbance of each fraction measured (manually) at 260 nm. Magnesium acetate (10 μl of 1 M) was added to each fraction to increase the concentration of magnesium ions to 20 mM. The fractions were then mixed with equal amounts of 100% of ice-cold ethanol, and their proteins precipitated overnight at -80°C. The precipitates were collected by centrifugation at 12,000 g for 30 min. The pellets were resuspended in 100 μl of buffer A. Forty μl of the suspension from each fraction was mixed with 10 μl 4× loading buffer and boiled, after which 25 μl of each sample was loaded onto each well for SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes, probed with anti-Obg antiserum, and the blots probed by ECL chemiluminescence method (Amersham). Association of Obg with ribosomal subunits was determined by comparing the immunoblot for each fraction with its absorbance at 260 nm.
Yeast two-hybrid assay
Protein-protein interactions were performed using the Matchmaker Gal4 two-hybrid system 3 (Clontech, Palo Alto, CA) as described previously . The yeast strain AH109, which has the reporter genes ADE2 (adenine), HIS3 (histidine), and MEL1 (α-galactosidase), was used as the host strain. Yeast plasmids (Table 2) were transformed into AH109 in appropriate combinations (Table 1) using standard protocols provided by Clontech. Expression of proteins by plasmids created for yeast two-hybrid analysis was assessed by the TNT Quick transcription and translation system (Promega), before transformation of the plasmids into yeast. Protein-protein interactions were determined by positive growth of yeast in synthetic drop out medium (SD) plates lacking adenine and histidine, and by the presence of blue color, which identifies α- galactosidase activity. To rule out false activation of the reporter gene, we transformed each of the constructs separately into yeast strain AH109, and assessed reporter gene activation. The strength of the interaction was verified by measuring the α-galactosidase released into the growth medium, again using protocols provided by Clontech.
SDS-PAGE and immunoblot
SDS-PAGE and immunoblotting were performed following the methods of Ausubel et al . Protein contents in extracts of E. coli or M. tuberculosis, obtained through sonication or bead-beating techniques, were determined by BCA (bicinchoninic acid) method (Pierce). Proteins were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with rabbit anti-M. tuberculosis Obg antiserum (1:500 dilution) or rabbit anti-M. tuberculosis SigH antiserum (1:1000), developed against recombinant His10-Obg or His10-SigH proteins, respectively. Alkaline phosphatase-conjugated anti-rabbit IgG (Zymed, 1:1000 dilution) or peroxidase-conjugated anti-rabbit IgG (Sigma, 1:10,000 dilution) were used as secondary antibodies. The blots were developed either with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate (Sigma, for alkaline phosphatase), or with an ECL kit (Amersham, for peroxidase).
This study was partly supported by Institutional Research Grant and San Antonio Area foundation.
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