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Acidic phospholipids have been shown to promote dissociation of bound nucleotides from Mycobacterium tuberculosis DnaA (DnaATB) purified under denaturing conditions (Yamamoto et. al, Biochemical J., 363, 305-311 (2002)). In the present study, we show that a majority of DnaATB in non-overproducing cells of M. tuberculosis is membrane associated. Estimation of phospholipid phosphorus following chloroform: methanol extraction of soluble DnaATB purified under native conditions (nDnaATB) confirmed the association with phospholipids. nDnaATB exhibited weak ATPase activity, and rapidly exchanged ATP for bound ADP in the absence of any added phospholipids. We suggest that the outcome of intracellular DnaATB -nucleotide interactions, hence DnaATB activity, is influenced by phospholipids.
Tuberculosis is one of the most globally prevalent infectious diseases and accounts for approximately three million deaths each year. The causative agent Mycobacterium tuberculosis, a Gram-positive bacterium, is a slow grower with approximate doubling time of 24 h. The genus Mycobacterium includes other pathogens such as M. tuberculosis, M. bovis, and M. leprae, and non-pathogens such as M. smegmatis and M. fortuitum. The doubling times of these organisms range from 2 to 3 h (M. smegmatis, M. fortuitum) to 22-24 h (M. tuberculosis, M. bovis) to 185 h (M. leprae). The genetic and biochemical factors responsible for the differences in the growth rates of various mycobacteria are largely unknown.
Chromosomal DNA replication in bacteria is regulated at the initiation step, where the activity and quantity of the initiator DnaA protein is critically controlled (1-3). DNA replication in Escherichia coli is initiated by the binding of DnaA protein to the DnaA boxes in the oriC, the origin of chromosomal DNA replication, and these initial interactions result in the melting of the nearby A-T-rich region, thereby forming an open (initiation) complex (4, 5). DnaA protein then recruits the DnaB helicase-DnaC protein complex to form a pre-priming complex, which allows entry of primase and establishment of the replication forks.
E. coli DnaA activity is proposed to be regulated by its binding of adenine nucleotides. DnaA protein has high affinity for ATP and ADP, and the ATP-binding form of DnaA protein (ATP-DnaA) initiates DNA replication in vitro, whereas the ADP-binding form (ADP-DnaA) does not (6). Membrane phospholipids are believed to play a crucial role in the regulation of DnaA activity (7). They affect the dissociation of both ATP and ADP from DnaA and rejuvenate moribund DnaA in to an active species for the oriC (8, 9). Acidic phospholipids in a fluid membrane facilitate dissociation of ADP bound to DnaA protein in vitro, and the resultant free form of DnaA protein is reactivated through binding to ATP, which is present at high concentrations under physiological conditions.
The key elements involved in the initiation of M. tuberculosis DNA replication, namely oriC and DnaA, have been identified (10, 11). M. tuberculosis oriC is 550-bp long (10) and recombinant DnaATB protein purified under denaturing conditions has been shown to associate with adenine nucleotides and oriC (12). DnaATB specifically recognizes DnaA boxes and dimethylsulfate footprinting revealed the presence of nine DnaA-boxes that bear little or no sequence similarity to the five DnaA boxes of E. coli oriC (13). Acidic phospholipids have been shown to modulate DnaATB interactions with adenine nucleotide and oriC stabilizes DnaATB-ATP interactions and promotes dissociation of DnaATB-ADP complexes (12). Presumably, the interaction of DnaATB with phospholipids results in a decrease in the affinity of DnaA for ATP, oriC or both. Other results suggest that ADP-bound form of DnaA is not competent for binding and rapid oligomerization on oriC (13). Together, these results suggest that phospholipids regulate nucleotide bound state of DnaA and play important regulatory roles in M. tuberculosis oriC replication (12). To further understand the relationships between DnaATB and phospholipids, we purified DnaATB as a soluble protein under native conditions and investigated its interactions with nucleotides in the absence of added phospholipids.
Cloning of M. tuberculosis dnaA was performed as described (12). Overproduction of the recombinant DnaA protein was carried out with co-producing E. coli thioredoxin (Trx) (14). For purification, bacterial pellets were collected and resuspended in Binding buffer [20 mM Tris/HCl (pH 8.0), 10 % glycerol, 500 mM NaCl, 5 mM 2-mercaptoethanol, 10 mM imidazole and 0.5 % Tween 20] and disrupted by sonication. The crude cell lysate was clarified by centrifugation, and the supernatant fraction, which contained the soluble recombinant DnaA protein, was loaded on a Ni2+-nitrilotriacetic acid-agarose column (Qiagen) equilibrated with same buffer. Unless otherwise noted, the purification was done at 4 °C. After washing with the same buffer to remove unbound proteins, DnaA protein was eluted with Renaturation buffer (50 mM Pipes (pH 6.8), 10 mM magnesium acetate, 200 mM ammonium sulfate, 20 % sucrose, 0.1 mM EDTA and 2 mM 2-mercaptoethanol) with 100 mM imidazole. Protein fractions were analyzed by SDS-PAGE (15). Fractions containing the eluted DnaA protein were pooled and dialyzed against Renaturation buffer. Aggregated protein was removed by centrifugation and the clear supernatant containing soluble DnaA protein was stored at -70 °C until use. Preparation of the denatured DnaA protein of M. tuberculosis with urea was performed according to our previous paper (12).
Nucleotide-binding experiments were carried out as explained (12). Binding of ATP to DnaA was performed in 100 μl of buffer E [50 mM Tris acetate (pH 8.2), 0.5 mM magnesium acetate, 0.3 mM EDTA, 5 mM 2-mercaptoethanol, 10 mM ammonium sulfate, 20 % glycerol, 0.005 % (v/v) Tween 20] containing various concentrations of [α-32P] ATP and DnaA protein (1 pmol). After incubation at 37 °C for 15 min, the reaction mixture was filtered through a nitrocellulose membrane (Millipore HA 0.45 μm, 24 mm diameter) presoaked in buffer E. The filter was washed with 10 ml of ice-cold buffer E, dried and the amount of radioactivity retained on the filter was measured in a liquid-scintillation counter. For exchange of ATP with ADP bound to DnaA protein, DnaA protein (2.0 pmol) was incubated with 200 pmol of [14C] ADP in 100 μl of buffer E at 0°C for 15 min, and then with 1.0 mM [α-32P] ATP at 37 °C. The reaction mixture was filtered through a nitrocellulose membrane and the amount of radioactivity retained on the filter was measured as described above.
ATPase activity of DnaA was measured as described (12). Reaction samples were prepared on ice in 40 μl of buffer E containing [α-32P]ATP (100 pmol) and DnaA protein (40 pmol) and transferred to 37 °C. Some samples contained the M. tuberculosis DnaA and adenine-nucleotide interactions M. tuberculosis oriC (0.2 μg). At indicated time intervals, samples were collected on membranes and bound nucleotides were extracted with 50 μl of 1 M formic acid. An aliquot (0.5 μl) of the extracted sample was spotted on the polyethyleneimine (PEI) cellulose (Macherey-Nagel) membranes and subjected to chromatography in 1 M formic acid-0.5 M LiCl. Membranes were dried, spots were visualized in a Molecular Imager (Bio-Rad) and quantified by using the Quantity One program.
The concentration of organic phosphate was estimated following Ames and Dublin protocol (16). Ten percent of magnesium nitrate (0.03 ml) was added to 0.1 ml of sample in Pyrex 13 × 100 mm test tube. The mixture was taken to dryness and ashed by shaking the tube over a strong flame. The tube was then allowed to cool and 0.3 ml of 0.5 N HCl is added. The material was heated in a boiling water for 15 min and cooled to room temperature. 0.7 ml of a solution containing 1.3 % ascorbic acid and 0.06 % Ammonium molybdate was added and the tube was incubated at 37 °C for 60 min. The color that has absorbancy at 820 nm was read and amount of phosphorous was estimated by comparing with standard prepared using inorganic phosphate.
Approximately 1 μg protein corresponding to whole cell lysate, membrane, cell walls and cytosolic fractions of M. tuberculosis obtained from Tuberculosis Vaccine Testing and Research Materials, Colorado State University, NIH contract NO1-AI 40091, was resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with affinity purified anti- DnaATB antibodies as described (17).
To test association, if any, of DnaATB protein with phospholipids, we probed whole cell lysates, soluble cell wall fractions, high-speed membrane and cytosolic fractions prepared from wild-type, DnaA non-overproducing M. tuberculosis cells with affinity purified anti- DnaATB antibodies. As can be seen, a majority of DnaATB protein in non-overproducing cells is membrane and cell wall associated, but hardly any in the cytosolic fractions (Fig. 1, compare lane 5 with lanes 2-4). These results are consistent with a notion that native DnaATB, like its E. coli counterpart, is associated with membranes and cell walls.
Membrane association of DnaATB indicate possible association with phospholipids. To further address this issue, we purified DnaATB under soluble conditions from E. coli cells expressing E. coli thioredoxin (Trx) and dnaA genes simultaneously. Coexpression of trx promoted the solubility of DnaA, consistent with other report on the increase in the solubility of various foreign proteins with Trx expression (14). As can be seen, a distinct band corresponding to DanA was evident (Fig. 2A). Clear cell lysates were then used to purify DnaATB on nickel affinity columns and SDS-PAGE analysis confirmed the purity of the soluble DnaA protein (nDnaATB) (Fig. 2A). Nucleotide-binding experiments revealed that purified nDnaATB bound ATP in protein concentration dependent manner, and that the amount bound was approximately 5-fold lower than that purified under denaturing conditions (Fig. 2B). Scatchard analysis revealed that KD for nDnaATB is approximately 415 nM (data not shown). This value is higher than those for DnaATB (69 nM) (12) and for E. coli DnaA protein (30 nM) (6).
Our results showing the association of DnaA with membrane fractions in non-overproducing cells (Fig. 1) and combined with reduced binding of DnaATB to ATP led us to hypothesize that nDnaATB is associated with phospholipids. To test this prediction, we extracted nDnaATB with chloroform: methanol (2: 1 v/v), and evaluated the presence of phospholipid phosphorus by colorimetric assay using inorganic phosphate as control. Our results indicate that amount of phospholipids bound to native DnaA was 1.87 pmol phospholipids / 7.4 pmol DnaA protein (data not shown). Since phospholipids promote dissociation of bound nucleotides, we predicted that the biochemical properties of the nDnaATB, i.e. ATPase activity and exchange of ATP to bound ADP, be different from that purified under denaturing conditions.
We detected the ATPase activity of the nDnaATB by PEI-thin layer chromatography. The DnaA protein showed a weak ATPase activity with turnover (catalytic-centre activity) of 7.5 × 10-4 (min-1). Similar results were obtained with the denatured DnaA, although the denatured DnaA exhibited an ATPase activity with turnover number of 0.016 (min-1) (12). It should be noted that the DnaA proteins of both E. coli and Bacillus subtilis also hydrolyze ATP slowly (6, 18). The slow turnover rate suggests that either the ATPase activity of M. tuberculosis DnaA is intrinsically low and or that the protein retains ADP following the hydrolysis of ATP. Determination of radiolabelled nucleotide retained by DnaA as a function of time showed a slow time-dependent decrease in bound ATP with a concomitant increase in bound ADP (Fig. 3A). Hydrolysis of the tightly bound ATP in the presence of oriC was 50 % complete in about 3 min whereas, in the absence of oriC, 50 % hydrolysis was achieved in about 10 min (Fig. 3A), suggesting that M. tuberculosis oriC stimulated the ATPase activity of the native DnaA. In case of the denatured DnaA, hydrolysis of bound ATP was 50 % complete in approx. 44 min without oriC, whereas it took 22 min in the presence of oriC (Fig. 3B).
The observed weak ATPase activity of DnaATB suggested the exchange of ATP for bound ADP is very low. To test this hypothesis, DnaA protein-[14C]ADP complexes were prepared first and then challenged with [α-32P]ATP. After incubation for different periods, samples were removed, filtered and the amount of [α-32P]ATP bound to DnaA was determined by scintillation counting. Parallel change experiments with non-radiolabelled ATP were carried out to determine the amount of ADP dissociated. It took approximately 10 min to exchange 50 % of the bound ADP for ATP (Fig. 4). On the other hand, seventy min was needed for the exchange with the denatured DnaA (12). The observed decrease in ATP-binding after 20 min (Fig. 4) is due to the fact that release of bound nucleotide from DnaA protein was promoted when purified under native conditions. With the native DnaA, exchange of ATP for bound ADP was also facilitated.
Considering the presence of phospholipid phosphorous in the chloroform-methanol extracted samples of nDnaATB (this study), and the ability of phospholipids to promote dissociation of bound nucleotides from DnaA purified under denaturing conditions (12), we reasons that DnaATB association with phospholipids could account for the observed differences in activities between the two protein preparations. It should be noted that solubilization of the DnaA with urea treatment was effective for the removal of any nucleotides and phospholipids associated with DnaA, much like the situation reported in E. coli (19). Phospholipids have been hypothesized to regulate DnaA activity of E. coli oriC replication (20). Presumably, this may well be the case with Mycobacteria, although the nature of the individual phospholipids that regulate DnaA activity may be different in both organisms. One characteristic feature of mycobacteria is their lipid-rich cell walls (21, 22). Phosphatidylinositol is one of major phospholipids of M. tuberculosis and it has been shown to be most effective agent for the dissociation of DnaA to ATP and ADP (12).
It is hypothesized that for the acidic phospholipid-mediated dissociation of adenine nucleotide bound to DnaA protein, electrostatic interactions followed by insertion of a part of the DnaA protein into the membrane is required (23). On the basis of two potential amphipathic helices in the E. coli DnaA protein (K327-I344 and D357-V374) responsible for interactions with acidic phospholipids (24, 25), we found a corresponding membrane binding domain in the M. tuberculosis DnaA protein that constitutes helix 1 (A393-D405) and sub-helices (D355-R367 and I369-A3810). Our results tend to suggest that the M. tuberculosis DnaA protein interacts with and possibly attaches to the cell membrane in vivo.
Ichihashi and his colleague reported that the inhibitory effect of basic or neutral phospholipid on the interaction of acidic phospholipids with DnaA protein in E. coli (23). The biochemical findings suggest that DnaA activity is regulated by a change in the phospholipid composition of the cell membrane under physiological conditions. Although this may be an essential feature of the regulation of M. tuberculosis oriC replication, the nature of the rejuvenation of M. tuberculosis DnaA remains uncertain. Considering the complex nature of mycobacterial cell membranes, further studies are required to examine the effects of phospholipids on the regulation of DnaA activities.
This work was supported in part by grants from the National Institutes of Health to MM (AI41406) and MR (AI48417).
Publisher's Disclaimer: Note: This is a pre-copy-editing, author-produced PDF of an article accepted for publication in the Journal of Biochemistry following peer review. The definitive publisher-authenticated version is at: Journal of Biochemistry 2008 143(6):759-764; doi:10.1093/jb/mvn025