B-subunit of Phosphate-specific Transporter fromMycobacterium tuberculosis Is a Thermostable ATPase

The B-subunit of phosphate-specific transporter (PstB) is an ABC protein. pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli. The overexpressed protein was found to be in inclusion bodies. The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography. The molecular mass of the protein was ∼31 kDa. The eluted protein showed ATP-binding ability and exhibited ATPase activity. Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M. tuberculosisPstB-ATPase. The study of the kinetics of ATP hydrolysis yieldedKm of ∼72 μm andVmax of ∼0.12 μmol/min/mg of protein. Divalent cation like manganese was inhibitory to the ATPase activity. Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme. The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5′-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a Ki of ∼0.40 mm. Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase. Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria. The B-subunit of phosphate-specific transporter (PstB) is an ABC protein. pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli. The overexpressed protein was found to be in inclusion bodies. The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography. The molecular mass of the protein was ∼31 kDa. The eluted protein showed ATP-binding ability and exhibited ATPase activity. Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M. tuberculosisPstB-ATPase. The study of the kinetics of ATP hydrolysis yieldedKm of ∼72 μm andVmax of ∼0.12 μmol/min/mg of protein. Divalent cation like manganese was inhibitory to the ATPase activity. Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme. The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5′-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a Ki of ∼0.40 mm. Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase. Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria. phosphate-specific transporter ATP-binding cassette adenosine triphosphatase 5′-p-fluorosulfonylbenzoyl adenosine immunoglobulin G isopropyl-β-d-thiogalactopyranoside polyacrylamide gel electrophoresis polymerase chain reaction inorganic phosphate B subunit of phosphate-specific transporter Importance of phosphate as an essential component of several biomolecules, such as membrane lipids, complex carbohydrates, nucleic acids, etc., is well known. Therefore, assimilation of phosphate from the environment and its metabolism are essential events for microorganisms for their survival. As phosphate is often a limiting nutrient, its import in bacteria is accomplished through several parallel transport systems (1Silver S. Walderhaug M. Microbiol. Rev. 1992; 56: 195-228Crossref PubMed Google Scholar, 2Braibant M. Gilot P. Content J. FEMS Microbiol. Rev. 2000; 24: 449-467Crossref PubMed Google Scholar). Phosphate-specific transporter (Pst)1 is one of them that has been reported to be present in several bacteria like Bacillus subtilis (3Qi Y. Kobayashi Y. Hulett F. J. Bacteriol. 1997; 179: 2534-2539Crossref PubMed Google Scholar), Escherichia coli (4Surin B.P. Rosenberg H. Cox G.B. J. Bacteriol. 1985; 161: 189-198Crossref PubMed Google Scholar, 5Wanner B.L. Neidhard F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low R.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. E. coli and Salmonella: Cellular and Molecular Biology. I. ASM Press, Washington, D. C.1996: 1356-1381Google Scholar),Mycobacterium tuberculosis (6Braibant M. Lefevre P. Dewit L. Oomes J. Peirs P. Hugyen K. Wattiez R. Content J. FEBS Lett. 1996; 394: 206-212Crossref PubMed Scopus (40) Google Scholar, 7Cole S.T. et al.Nature. 1998; 395: 537-544Crossref Scopus (6660) Google Scholar), Salmonella typhimurium (5Wanner B.L. Neidhard F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low R.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. E. coli and Salmonella: Cellular and Molecular Biology. I. ASM Press, Washington, D. C.1996: 1356-1381Google Scholar), Streptococcus pneumoniae (8Novak R. Cauwels A. Charpentier E. Tuomanen E. J. Bacteriol. 1999; 181: 1126-1133Crossref PubMed Google Scholar), etc. Pst is a tightly regulated high affinity system grouped under ATP-binding cassette (ABC) transporters, which includes the largest family of paralogous proteins that are present in wide variety of cells including those of mammals (9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3425) Google Scholar, 10Doige C.A. Ames G.F. Annu. Rev. Microbiol. 1993; 47: 291-319Crossref PubMed Scopus (269) Google Scholar, 11Gottesman M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3590) Google Scholar). Expression of Pst is operon-controlled, and the import function of this multisubunit transporter is known to be operative only during phosphate limitations (see Ref. 5Wanner B.L. Neidhard F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low R.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. E. coli and Salmonella: Cellular and Molecular Biology. I. ASM Press, Washington, D. C.1996: 1356-1381Google Scholar and references therein). Besides transporting phosphate, the Pst system in bacteria has also shown to be involved in controlling a number of co-ordinately regulated genes, grouped under the pho regulon (5Wanner B.L. Neidhard F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low R.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. E. coli and Salmonella: Cellular and Molecular Biology. I. ASM Press, Washington, D. C.1996: 1356-1381Google Scholar). Interestingly, among the available prokaryotic genome sequences, only in M. tuberculosis have three putative pstoperons been identified (6Braibant M. Lefevre P. Dewit L. Oomes J. Peirs P. Hugyen K. Wattiez R. Content J. FEBS Lett. 1996; 394: 206-212Crossref PubMed Scopus (40) Google Scholar, 7Cole S.T. et al.Nature. 1998; 395: 537-544Crossref Scopus (6660) Google Scholar). Therefore, it has been thought that many copies of the same phosphate transporter in mycobacteria might be involved in subtle biochemical adaptations of this microorganism for its growth and survival under highly varying (e.g.phosphate-limiting) conditions during infectious cycle (2Braibant M. Gilot P. Content J. FEMS Microbiol. Rev. 2000; 24: 449-467Crossref PubMed Google Scholar). Besides phosphate transport, the role of this transporter in coping up with adverse situations in mycobacteria has also been postulated (6Braibant M. Lefevre P. Dewit L. Oomes J. Peirs P. Hugyen K. Wattiez R. Content J. FEBS Lett. 1996; 394: 206-212Crossref PubMed Scopus (40) Google Scholar,12Lefevre P. Braibant M. Dewit L. Kalai M. Roeper D. Grotzinger J. Delville J. Peirs P. Oomes J. Hugyen K. Content J. J. Bacteriol. 1997; 179: 2900-2906Crossref PubMed Scopus (79) Google Scholar, 13Banerjee S.K. Misra P. Bhatt K. Chakraborti P.K. Mol. Gen. Genet. 2000; 262: 949-956Crossref PubMed Scopus (35) Google Scholar, 14Bhatt K. Banerjee S.K. Chakraborti P.K. Eur. J. Biochem. 2000; 267: 4028-4032Crossref PubMed Scopus (36) Google Scholar). In most of the prokaryotes, Pst is found as a membrane-associated complex. In E. coli it is composed of four distinct subunits encoded by pstS, pstA, pstC, andpstB genes (4Surin B.P. Rosenberg H. Cox G.B. J. Bacteriol. 1985; 161: 189-198Crossref PubMed Google Scholar) and arranged in an operon aspstSCAB (5Wanner B.L. Neidhard F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low R.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. E. coli and Salmonella: Cellular and Molecular Biology. I. ASM Press, Washington, D. C.1996: 1356-1381Google Scholar, 15Higgins C.F. Hiles I.D. Salmond G.P.C. Gill D.R. Downie J.A. Evans I.J. Holland I.B. Gray L. Buckel S.D. Bell A.W. Hermodson M.A. Nature. 1986; 323: 448-450Crossref PubMed Scopus (527) Google Scholar). PstS is the periplasmic binding protein. The PstA and PstC are integral membrane channel proteins and are hydrophobic in nature. PstB subunit, which is often referred as ABC protein (16Hunke S. Schneider E. FEBS Lett. 1999; 448: 131-134Crossref PubMed Scopus (12) Google Scholar), provides energy for transport through ATP hydrolysis (5Wanner B.L. Neidhard F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low R.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. E. coli and Salmonella: Cellular and Molecular Biology. I. ASM Press, Washington, D. C.1996: 1356-1381Google Scholar). Available reports indicated the similar organization of the genes of the pst operon in other prokaryotes (17Linton K.J. Higgins C.F. Mol. Microbiol. 1998; 28: 5-13Crossref PubMed Scopus (366) Google Scholar, 18Quentin Y. Fichant G. Denizot F. J. Mol. Biol. 1999; 287: 467-484Crossref PubMed Scopus (164) Google Scholar) as well, except for mycobacteria (7Cole S.T. et al.Nature. 1998; 395: 537-544Crossref Scopus (6660) Google Scholar, 19Cole S.T. et al.Nature. 2001; 409: 1007-1011Crossref PubMed Scopus (1389) Google Scholar). In M. tuberculosis, the presence of several copies of all the components of the operon except for PstB has been reported (6Braibant M. Lefevre P. Dewit L. Oomes J. Peirs P. Hugyen K. Wattiez R. Content J. FEBS Lett. 1996; 394: 206-212Crossref PubMed Scopus (40) Google Scholar, 7Cole S.T. et al.Nature. 1998; 395: 537-544Crossref Scopus (6660) Google Scholar). Bacterial ABC proteins have been shown to be responsible for ATP binding as well as ATP hydrolysis, which is evident from the studies with histidine and maltose transporters from S. typhimurium(16Hunke S. Schneider E. FEBS Lett. 1999; 448: 131-134Crossref PubMed Scopus (12) Google Scholar, 20Morbach S. Tebbe S. Schneider E. J. Biol. Chem. 1993; 268: 18617-18621Abstract Full Text PDF PubMed Google Scholar, 21Nikaido K. Liu P. Ames G.F. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In fact, structural data with the ATP-binding component of the S. typhimurium histidine permease corresponded well with the biochemical studies (22Hung Li. Wang I. Nikaido K. Liu P. Nature. 1998; 396: 703-707Crossref PubMed Scopus (629) Google Scholar). Even the homologous component of the Pst in E. coli has already been shown to possess ATP hydrolyzing ability (23Chan F.Y. Torriani A. J. Bacteriol. 1996; 178: 3974-3977Crossref PubMed Scopus (49) Google Scholar). The ATP-binding subunit of the bacterial ABC transporters have also been implicated in diverse biological functions (24Hiles I.D. Gallagher M.P. Jamieson D.J. Higgins C.F. J. Mol. Biol. 1987; 195: 125-142Crossref PubMed Scopus (188) Google Scholar, 25Dean D.A. Reizer J. Nikaido H. Saier M.H.Jr. J. Biol. Chem. 1990; 265: 21005-21010Abstract Full Text PDF PubMed Google Scholar, 26Panagiotidis C.H. Reyes M. Sievertsen A. Boos W. Shuman H.A. J. Biol. Chem. 1993; 268: 23685-23696Abstract Full Text PDF PubMed Google Scholar). We have reported previously that pstB is overexpressed as well as amplified in a fluoroquinolone-resistant colony of Mycobacterium smegmatis, suggesting a novel role of this subunit in addition to its involvement in the process of phosphate import (13Banerjee S.K. Misra P. Bhatt K. Chakraborti P.K. Mol. Gen. Genet. 2000; 262: 949-956Crossref PubMed Scopus (35) Google Scholar, 14Bhatt K. Banerjee S.K. Chakraborti P.K. Eur. J. Biochem. 2000; 267: 4028-4032Crossref PubMed Scopus (36) Google Scholar, 27Banerjee S.K. Misra P. Bhatt K. Mande S.C. Chakraborti P.K. FEBS Lett. 1998; 25: 151-156Crossref Scopus (17) Google Scholar). Furthermore, among all the prokaryotic genome sequences available so far, pstB has been found to be present throughout, giving strong indications that this gene might be important for the microorganisms. We therefore focused our effort to gain an insight on the nature of the mycobacterial PstB protein. In this article, we report that, unlike other prokaryotic ABC proteins, the ATP hydrolyzing ability of PstB from M. tuberculosis is rather magnesium-independent and resistant to known ATPase inhibitors. Furthermore, our results convincingly established that the mycobacterial PstB is a thermostable ATPase and thus highlighted the presence of such an enzyme in any mesophilic bacteria. Restriction/modifying enzymes and other molecular biological reagents were obtained from either New England Biolabs or Promega Corp. ATP-binding protein detection kit (Roche Molecular Biochemicals), ECL Western blotting detection kit (Amersham Pharmacia Biotech), Expand high fidelity PCR system (Roche Molecular Biochemicals), plasmid preparation kits (Qiagen), protein molecular weight markers (Sigma), x-ray film (Eastman Kodak) were commercially available. All other chemicals including urea, Triton X-100, guanidine hydrochloride, N-lauroylsarcosine (Sarkosyl), etc. were procured from Sigma. All oligonucleotides used in this study were custom synthesized from PMK International. [α-32P]dCTP was supplied by Jonaki Laboratories, BRIT, Hyderabad, India. The forward (CS1, 5′-CATATGGCGTGTGAACGGCTC-3′) and reverse (CS2, 5′-CTTTCTGAGCTCTTCAATT-3′) primers for PCR amplification ofpstB (Rv0933) were designed on the basis of the publishedM. tuberculosis genome sequence (7Cole S.T. et al.Nature. 1998; 395: 537-544Crossref Scopus (6660) Google Scholar). CAT in primer CS1 does not correspond to the genome sequence. It was introduced in CS1 to incorporate a NdeI site at the 5′ end of the amplified PCR fragment. Genomic DNA from M. tuberculosis H37Rv (obtained as a gift from Dr. Jaya Tyagi, Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India) was used as the template in Expand high fidelity PCR system (Roche Molecular Biochemicals). PCR was carried out for 30 cycles (denaturation at 94 °C for 30 s per cycle, annealing at 50 °C for 30 s per cycle, and elongation at 68 °C for 1 min for first 10 cycles; for the remaining 20 cycles, the elongation step was extended for an additional 5 s in each cycle). Following treatment with Klenow, the blunt-ended pstB was cloned at the EcoRV site of pBluescript (SK+) and the construct was designated as pCJS1. The nucleic acid sequence of the pstB was confirmed using an automated sequencer (ABI, PerkinElmer Applied Biosystems). The pstB fragment from pCJS1 was excised following restriction digestions with NdeI and BamHI (the enzyme site is absent in pstB but present in the vector and located at 3′ end after the stop codon) and subcloned at the corresponding sites in pET 23a (28Studier O.F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6225) Google Scholar). Following transformation inE. coli strain DH5α, plasmid DNA was extracted and the construct (pCJS2) was verified by restriction digestions. Genomic DNA from E. coli strain K12 (MTCC 1302) was also extracted following standard procedures (29Sambrook J. Fritsch E.F. Manitis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). For PCR amplification ofpstB from E. coli, the primers (CS3, 5′-GATTGCATATGAGTATG-3′; CS4, 5′-GAGACTGTCCATAACGCA-3′) were designed based on the published sequence (4Surin B.P. Rosenberg H. Cox G.B. J. Bacteriol. 1985; 161: 189-198Crossref PubMed Google Scholar) and the same strategy was adopted for cloning (pCRC2) into expression vector. PCR was employed to generate D188K (aspartic acid is replaced by lysine at amino acid residue 188) mutant in the Walker B motif (30Walker J. Saraste M. Runswick M. Gray N. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4401) Google Scholar) of the M. tuberculosis PstB. Two forward (CS1 and CS9) and two reverse primers (CS19 and CS21) were used for this purpose. Primers (CS9, 5′-GTTGCTGCTCAAGGAGCCCACC-3′; CS19, 5′-ACTTCAATTTCCGCGCTTGGC-3′; and CS21, 5′-GGTGGGCTCCTTGAGCAGCAAC-3′) were designed based on M. tuberculosis pstB sequences and base mismatches (underlined) were incorporated to obtain desired mutations. To generate the mutant, two sets of primary and one set of secondary PCR reactions were carried out (31Rana S. Bisht D. Chakraborti P.K. J. Mol. Biol. 1999; 286: 669-681Crossref PubMed Scopus (7) Google Scholar) using the gel-purified wild type pstB(831 base pairs) as template. Primary reactions were carried out with primers CS1/CS21 and CS9/CS19, whereas for secondary PCR reaction CS1 and CS19 were used. Thus, D188K mutation was contained within the amplified fragment of the PstB. Secondary PCR product was restriction-digested with AatII, which yielded a 655-base pair fragment containing the desired mutation, and finally substituted for the corresponding wild type fragment cloned in expression vector. Mutations were confirmed by sequencing. The pCJS2 was transformed into E. coli strain BL21(DE3) and selected on LB-ampicillin plates (100 μg/ml). Overnight cultures (∼15 h at 37 °C) of several colonies were reinoculated and grown untilA600 reached ∼0.45. Cultures were then induced with 0.4 mmisopropyl-β-d-thiogalactopyranoside (IPTG). Cells were harvested after 2 h, lysates were prepared, and expression was checked by running 12% SDS-PAGE, followed by Coomassie Brilliant Blue staining. To know the solubility of the expressed protein, cells after induction were suspended in lysis buffer (100 mm Tris, pH 7.5, containing 5 mm EDTA, 5 mm dithiothreitol, and 5 mm phenylmethylsulfonyl fluoride), treated (20 min at 24 °C) with lysozyme (200 μg/ml), and sonicated, and different fractions (supernatant and pellet after centrifugation at 22,000 × g for 30 min at 4 °C) were subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining. Pellet fractions obtained following centrifugation of IPTG-induced sonicated cells (transformed either with pCJS2 or pCRC2) were washed in wash buffer (lysis buffer supplemented with 4 m urea and 5% Triton X-100) following the procedure described elsewhere (32Coligan J.E. Dunn B.M. Ploegh H. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. John Wiley & Sons, New York1995Google Scholar). The washed pellets (inclusion bodies containing expressed protein) were suspended in TEN buffer (10 mm Tris, 1 mm EDTA, and 150 mm NaCl, pH 7.5) containing 1.5% Sarkosyl, and were subjected to ultracentrifugation (107,000 × g for 1 h at 4 °C in an ultracentrifuge, Beckman). The supernatant fractions collected in this way contained solubilized protein and were subjected to gel permeation chromatography in a fast protein liquid chromatography unit (Amersham Pharmacia Biotech) using Superdex 200 column. Protein was eluted with TEN buffer containing 0.15% Sarkosyl. The inclusion body containing PstB protein fromM. tuberculosis was solubilized using buffer (50 mm Tris, 5 mm EDTA, and 5 mmdithiothreitol, pH 7.5) containing guanidine hydrochloride (8m) following standard procedures (32Coligan J.E. Dunn B.M. Ploegh H. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. John Wiley & Sons, New York1995Google Scholar). The denatured protein obtained in this way (purity ∼85%, as evidenced by SDS-PAGE) was precipitated by removing guanidine hydrochloride through dialysis (buffer: 50 mm Tris, 5 mm EDTA, 5 mm dithiothreitol, and 1 m NaCl, pH 7.5) and was used to raise polyclonal antibodies in rabbit. Briefly, purified protein (∼800 μg) emulsified in complete Freund's adjuvant was injected subcutaneously at multiple sites. Boosters were emulsified in incomplete Freund's adjuvant and were given at the same dose at intervals of 21 days. After the third booster dose, blood was collected and sera prepared was decomplimented at 56 °C for 30 min. The antibody titer was determined by indirect enzyme-linked immunosorbent assay using horseradish peroxidase-conjugated anti-rabbit IgG as secondary antibody and 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) as substrate. The antisera showed cross-reactivity to PstB fromE. coli. Similarly, antibody raised against E. coli PstB (obtained as a gift from Dr. A. Torriani, Massachusetts Institute of Technology, Cambridge, MA) recognized mycobacterial PstB. ATP-binding ability of the recombinant PstB was monitored by labeling the protein with a nonhydrolyzable ATP analogue, 5′-p-fluorosulfonylbenzoyl adenosine (FSBA). For the labeling reaction, the recombinant PstB protein (dissolved in borate buffer, pH 7.4, supplemented with 0.15% Sarkosyl) was incubated (30 min at 30 °C) with 1–3 mmFSBA. The samples were run on SDS-PAGE, followed by detection through Western blotting using anti-FSBA antibody. The ATPase activity of the protein was quantitated by a colorimetric assay performed in microtiter plates following the method described by Henkel et al. (33Henkel R. Vandeberg J. Walsh A. Anal. Biochem. 1988; 169: 312-318Crossref PubMed Scopus (114) Google Scholar). Briefly, PstB protein (0.5 to 1 μg) was diluted with TEN buffer to a final volume of 25 μl. The reaction was initiated by adding equal volume of substrate solution (ATP final concentration = 1 mm; unless mentioned otherwise), followed by incubation at 37 °C for 5 min (final Sarkosyl concentration = 0.06%). The enzymatic reaction was terminated by addition of an acidic solution (200 μl) of malachite green, ammonium molybdate, and polyvinyl alcohol (33Henkel R. Vandeberg J. Walsh A. Anal. Biochem. 1988; 169: 312-318Crossref PubMed Scopus (114) Google Scholar). The activity was measured as the amount of inorganic phosphate (Pi) liberated that forms a phosphomolybdate malachite complex detected at 650 nm in an enzyme-linked immunosorbent assay plate reader. The values obtained were corrected by subtracting the blank readings obtained for nonenzymatic release of Pibecause of hydrolysis of ATP and Pi contamination in the absence of enzyme as well as substrate. A standard curve with sodium phosphate monobasic was run concurrently with each experiment, and thus nanomoles of Pi released were calculated. ATPase activity is expressed as nanomoles of Pi liberated/min/mg of protein, and the data presented in the form of mean ± S.D. EDTA was omitted from the Sarkosyl-supplemented TEN buffer in carrying out studies with divalent cations. The effect of different divalent cations (Ca2+, Mg2+, and Mn2+) on the ATPase activity was monitored by adding them in the presence or absence of 5 mm EDTA prior to incubation with substrate solution. Different inhibitors used in this study were sodium azide,N-ethylmaleimide, and sodium orthovanadate. All inhibitors were dissolved in assay buffer, except for N-ethylmaleimide, which was in ethanol. To elucidate the effect ofN-ethylmaleimide, final concentration of ethanol during the assay was adjusted to 0.6% and accordingly proper blank was maintained. The influence of inhibitors was determined by incubating (15 min or 3 h at room temperature) them with the protein prior to the addition of substrate solution. Effect of temperature (80 °C) for different time periods (0–60 min) on aggregation pattern of PstB proteins from M. tuberculosis and E. coli in solution (protein concentration of ∼100 μg/ml in TEN buffer containing 0.06% Sarkosyl) was examined by studying Raileigh's scattering at 600 nm (λexcitation = λemission) in a fluorometer (PerkinElmer Life Sciences). To confirm cloning of PCR products in vectors, Southern hybridization was carried out following standard protocols (29Sambrook J. Fritsch E.F. Manitis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) using [α-32P]dCTP-labeled probes. Western blotting was employed to examine the expression of PstB protein or to detect FSBA-bound protein. Protein was estimated following Bradford's method (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222415) Google Scholar). Purified proteins or cell extracts (800 ng to 3 μg protein/slot) were resolved in SDS-PAGE and transferred at 100 V for 45 min to nitrocellulose membrane (0.45 μm) in a mini-transblot apparatus (Bio-Rad) using Tris-glycine buffer (48 mm Tris, 39 mm glycine, 0.037% SDS, and 20% methanol, pH ∼8.3). Blots were probed with primary (anti-PstB or anti-FSBA) and secondary (horseradish peroxidase-conjugated anti-rabbit IgG) antibodies and processed with ECL detection system as described elsewhere (31Rana S. Bisht D. Chakraborti P.K. J. Mol. Biol. 1999; 286: 669-681Crossref PubMed Scopus (7) Google Scholar). Stripping of the blots, if necessary, was done following the manufacturer's recommended protocol (Amersham Pharmacia Biotech). The pstB gene fromM. tuberculosis strain H37Rv was amplified by PCR.pstB-specific primers (CS1 and CS2) were designed based on the published M. tuberculosis genome sequence (7Cole S.T. et al.Nature. 1998; 395: 537-544Crossref Scopus (6660) Google Scholar). PCR was carried out at annealing temperature of 50 °C with primers and genomic DNA utilizing a mixture of Taq and PwoDNA polymerase, which resulted in the amplification of expected ∼831-base pair fragment. Only those reactions, which contained template DNA, primers, and enzymes, showed the amplification (data not shown). The PCR-amplified fragment was cloned in pBluescript (SK+) and was sequenced. A base pair change was observed at codon 235. However, it did not result in alteration of any amino acid because codon 235, AAG (coding for phenylalanine), was altered to AAA. PstB was overexpressed following subcloning in pET23a (see "Experimental Procedures"). Several colonies showed the overexpression of the protein, as evidenced by an expected band of ∼31 kDa in SDS-PAGE following staining with Coomassie Brilliant Blue in cultures transformed with pCJS2 and induced with IPTG. One of these colonies was selected for further processing (Fig.1). However, the expressed protein was observed in the pellet fraction (inclusion bodies) in SDS-PAGE analysis (Fig. 1, lane 4). Cultures grown at even lower temperatures did not yield any soluble protein. Such an event is not restricted to PstB from M. tuberculosis only because homologous protein from E. coli when expressed following transformation of pCRC2 was also found to be in inclusion bodies (data not shown). PstB aggregates were partially solubilized with 1.5% Sarkosyl (Fig. 1, compare lanes 6 and 7) and were purified by gel permeation chromatography. Column elutes formed a single peak within the separation range of Superdex 200, SDS-PAGE analysis of which is also shown in Fig. 1 (lanes 8 and 9). The molecular weight of the eluted protein obtained through gel permeation chromatography was found to be 69.3 ± 3.25 (mean ± S.D.,n = 7), whereas the same samples subjected to SDS-PAGE revealed a molecular mass of 30.5 ± 1 kDa (mean ± S.D.,n = 5). Western blot with the anti-PstB antibody recognized the purified protein (Fig. 2). Thus our data argue that the active form of the PstB from M. tuberculosis is possibly a dimer. This is not unusual because the nucleotide-binding subunits of bacterial ABC transporters are known to be active as dimer (21Nikaido K. Liu P. Ames G.F. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 23Chan F.Y. Torriani A. J. Bacteriol. 1996; 178: 3974-3977Crossref PubMed Scopus (49) Google Scholar, 35Webb D. Rosenverg H. Cox G. J. Biol. Chem. 1992; 267: 24661-24668Abstract Full Text PDF PubMed Google Scholar).Figure 2ATP-binding ability of mycobacterial PstB. PstB following incubation with different concentrations of FSBA were subjected to SDS-PAGE and immunoblotting using anti-FSBA (aFSBA) or anti-PstB (aPstB) antibodies.Lane 1, PstB with no FSBA; lane 2, PstB with 1 mm FSBA; lane 3, PstB with 3 mmFSBA.View Large Image Figure ViewerDownload (PPT) Nucleotide-binding subunits of different bacterial ABC transporters including PstB from E. coli have been shown to bind ATP (23Chan F.Y. Torriani A. J. Bacteriol. 1996; 178: 3974-3977Crossref PubMed Scopus (49) Google Scholar, 36Walter C. Bentrup K. Schneider E. J. Biol. Chem. 1992; 267: 8863-8869Abstract Full Text PDF PubMed Google Scholar). To gain insight on this aspect, we utilized the binding ability of FSBA at the nucleotide binding sites of such proteins through covalent modification (37Anostario M. Harrison M. Geahlen R. Anal. Biochem. 1990; 190: 60-65Crossref PubMed Scopus (22) Google Scholar). Following labeling of protein (purified in borate buffer) with FSBA (1 or 3 mm) or treating with Me2SO (solvent control), samples were subjected to SDS-PAGE and immunoblotting using anti-FSBA antibody. As shown in Fig. 2 A, anti-FSBA antibody recognized only those samples that were incubated with FSBA (lanes 2 and 3). On the other hand, the same blot, following stripping, when probed with anti-PstB antibody, rec