Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany
Correspondence
Erwin Schneider
erwin.schneider{at}rz.hu-berlin.de
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ABSTRACT |
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Present address: Max-Delbrück-Center Berlin-Buch, Robert-Rössle-Str. 10, 13092 Berlin, Germany.
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INTRODUCTION |
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ABC transporters mediating the uptake of amino acids have been classified in three separate subfamilies designated PAAT (polar amino acid transporters), HAAT (hydrophobic amino acid transporters) and MUT (methionine uptake transporters) (Hosie & Poole, 2001; Saier, 2000
; Zhang et al., 2003
). The PAAT family comprises most of the known transporters, including the well-studied histidine permease of Salmonella enterica serovar Typhimurium (S. typhimurium) (reviewed by Schneider, 2003
). However, very few of the other family members are functionally characterized. In particular, this holds true for transport systems from thermophilic micro-organisms. For example, in the Gram-positive bacterium Bacillus (now Geobacillus) stearothermophilus NUB36, which grows best at 5560 °C, only two genes have been characterized so far, the products of which are involved in glutamine uptake (Wu & Welker, 1991
). The proteins displayed significant sequence identities to the ABC subunits and solute-binding proteins, respectively, of glutamine, histidine or arginine transport systems in Escherichia coli/S. typhimurium. This observation is consistent with the result of computer-aided analyses of complete genome sequences that group ABC transporters for glutamine and basic amino acids in one subfamily (Dassa & Bouige, 2001
). Thus, in order to obtain a more complete overview of the repertoire of ABC transporters for polar amino acids in G. stearothermophilus, we searched the incomplete genomic sequence of strain DSMZ 13240 (see http://www.genome.ou.edu/bstearo.html) by BLAST (at http://www.ncbi.nlm.nih.gov/) using the ATPase subunit HisP of the histidine/lysine-arginine-ornithine transporter (LAO/HisJ-QMP) of S. typhimurium (Schneider, 2003
) as the seed sequence. The data revealed highest identity (52 % compared to 37 % for the next best hit) to a putative ABC protein (27 kDa) encoded within contig 450 (the numbering is still subject to change). The corresponding gene is part of a cluster that additionally includes ORFs for a putative solute-binding protein (29 kDa) and a membrane-integral (permease) subunit (24 kDa). All three gene products also displayed similar identities to the corresponding components of the arginine (ArtPIQMJ) (Wissenbach et al., 1995
) and glutamine (GlnHPQ) (Nohno et al., 1986
) transporters of E. coli and to an arginine transporter (YqiXYZ) of Bacillus subtilis (Sekowska et al., 2001
). However, attempts to more precisely predict the substrate by PSI-BLAST searches gave contradicting results. While the ATPase and membrane-integral subunits displayed highest similarities to glutamine transport components, the solute-binding protein was predicted to be most similar to the arginine-binding protein (ArtJ) of E. coli (Wissenbach et al., 1995
). Thus, in order to clearly identify the physiological substrate(s) we have cloned and overexpressed the genes in E. coli and characterized the functions of their purified products by binding assays and in a reconstituted system. The results show that the gene cluster encodes a high-affinity arginine ABC transporter with low affinities for lysine and ornithine. In accordance with the E. coli nomenclature and supported by sequence alignments the genes are designated artJMP.
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METHODS |
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Standard DNA techniques.
Isolation of genomic DNA of G. stearothermophilus, preparation of plasmids, digestion by endonucleases, ligation reactions and PCR were performed as described previously (Hülsmann et al., 2000).
Cloning of artJ.
The artJ gene was amplified by PCR using the Taq platinum polymerase (Invitrogen), and G. stearothermophilus chromosomal DNA as the template. The primers (5'-CGC TCC TCG AGG CGG GCG GCA AGT CAA CGG AAA CAA G-3'; 5'-GAT CCA GAA TTC TCA CCT TTC TCT ACG AAA AAT GCG TAA CTT GC-3') were designed in such a way that the amplified fragment lacked the 5' sequence encoding the signal peptide and that in the translated mature polypeptide, alanine substituted for the N-terminal cysteine residue. The amplified fragment was ligated with pGEM-T, resulting in plasmid pAW10. To overexpress artJ in E. coli the gene was further subcloned (as an XhoIEcoRI fragment) into expression vector pBAD/HisA. The resulting plasmid was designated pAW11.
Cloning of artMP.
The artMP genes were co-amplified by PCR as one fragment. The primers (5'-AGA ACG GGA TCC GAT TTT CGT TTT GAT ATT ATT GTC-3'; 5'-GAG TGA GAT CTA AAT ACT TTT GAC AAA AAC GCT TTT G-3') were designed in such a way that the start codon of artM was replaced by a BamHI site and that a BglII site substituted for the stop codon of artP. The resulting 1·4 kb fragment was cloned into expression vector pQE60, yielding plasmid pRF2.
Purification of ArtJ.
E. coli strain TOP10(pAW11) was grown in LB, containing ampicillin, to an OD650 of 0·5 and supplemented with 0·02 % arabinose to induce artJ expression, and growth was continued for 4 h. Cells were harvested, resuspended in 50 mM MOPS/KOH, pH 7, 100 mM NaCl, 0·1 mM PMSF and disintegrated by one passage through a French press at 14 000 p.s.i. (96·6 MPa). After ultracentrifugation (45 min at 200 000 g), ArtJ was recovered in the supernatant and subsequently purified by immobilized metal-affinity chromatography using a Co2+-loaded resin (TALON, Clontech).
Purification of ArtMP.
E. coli strain JM109(pRF2) was grown under the same conditions as TOP10(pAW11) but adding 0·5 mM IPTG for induction of gene expression. For purification of ArtMP, the membrane fraction obtained after cell disruption and ultracentrifugation (1 h at 200 000 g) was resuspended in 50 mM Tris/HCl, pH 8, 5 % (v/v) glycerol, 0·1 mM PMSF and solubilized with 1·2 % decanoylsucrose. After ultracentrifugation (30 min at 200 000 g) the supernatant was adjusted to 10 mM ATP and 20 mM 2-mercaptoethanol and purified on a Co2+-loaded affinity matrix in the presence of 0·2 % decanoylsucrose.
Isolation of total lipids from G. stearothermophilus.
Lipids were extracted from cells of G. stearothermophilus by a published method (Folch et al., 1957). Briefly, cells (10 g wet weight) were washed once with 50 mM Tris/HCl, pH 8, homogenized in 20 vols chloroform/methanol (2 : 1, v/v) and stirred for 18 h at room temperature. Undissolved material was removed by filtration and the filtrate was extracted with 0·2 vols double-distilled water. After separation into two phases, the lower (organic) phase was evaporated to dryness and washed with acetone for 1 h at room temperature. After removing acetone with a pipette, the sediment was dried under a stream of nitrogen, dissolved to 20 mg ml1 in chloroform and stored at 20 °C.
Preparation of proteoliposomes.
The ArtMP complex was incorporated into liposomes essentially as described previously (Scheffel et al., 2004). Lipids (20 mg) were dried under a stream of nitrogen, and slowly redissolved in 50 mM MOPS/KOH, pH 7·5, containing 1 % octyl
-D-glucopyranoside. The mixture was then sonicated for 15 min, and then ArtMP (50 µg), ArtJ (120 µg) and L-arginine (1 mM, final concentration) were added to the lipid/detergent mixture. These values correspond to a 7·6-fold molar excess of binding protein over transport complex, which is in the range of what has been proven optimal for the maltose and histidine ABC transporters (Landmesser et al., 2002
; Hall et al., 1998
; Liu et al., 1997
). Proteoliposomes were formed by removal of detergent by adsorption to Biobeads (100 mg; Bio-Rad) at 4 °C overnight. After replacing the beads with a new batch incubation was continued for 1 h. The mixture was subsequently centrifuged for 1 min at 300 g to pellet the beads and the supernatant was assayed for ATPase activity. As controls proteoliposomes were prepared by the same procedure but omitting ArtJ/L-arginine or L-arginine.
Binding assay.
Binding of L-[14C]arginine to purified ArtJ was performed by the method of Richarme & Kepes (1983). Standard assay mixtures (100 µl) containing 50 mM MOPS/KOH, pH 7·5, 150 mM NaCl, 5 % (v/v) glycerol and purified ArtJ (3 µg) were preheated at 60 °C for 1 min prior to the addition of L-[U-14C]arginine (Hartmann Analytic; 264 mCi mmol1, 9·77 GBq; 5 µM final concentration). After 1 min, the reaction was terminated by adding 2 ml of an ice-cold saturated (NH4)2SO4 solution and immediately filtered through a nitrocellulose filter (0·45 µm). The filter was washed once with the same solution, followed by distilled water, and retained radioactivity was determined in a liquid scintillation counter. In competition experiments, unlabelled L-amino acids (0·5 mm) were added 1 min prior to the addition of L-[14C]arginine. To determine the binding constant, ArtJ was first subjected to a denaturation/renaturation procedure using guanidine hydrochloride (6 M) to remove excess bound unlabelled substrate (Hall et al., 1998
).
Analytical methods.
Hydrolysis of ATP was assayed in microtitre plates essentially as described by Landmesser et al., (2002). Protein was assayed by using the BCA kit from Bio-Rad. SDS-PAGE and immunoblot analyses were performed as described by Hülsmann et al. (2000)
.
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RESULTS AND DISCUSSION |
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ABC transporter functions of ArtJMP were subsequently studied in proteoliposomes by assaying the following well-established criteria: (i) stimulation of ATPase activity of the complex by the cognate binding protein (Davidson et al., 1992; Liu et al., 1997
; Landmesser et al., 2002
; Scheffel et al., 2004
), and (ii) sensitivity of this activity to the specific inhibitor orthovanadate (Landmesser et al., 2002
; Sharma & Davidson, 2000
). To this end, proteoliposomes were prepared from purified ArtMP and a crude total lipid extract of G. stearothermophilus (mainly composed of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol; Jurado et al., 1991
) in the presence of L-arginine (1 mM) and/or ArtJ. By this procedure, substrate and binding protein were enclosed in the lumen of the proteoliposomes. Assuming that incorporation of complex molecules occurs randomly with respect to orientation, as has been demonstrated for example for the maltose and histidine ABC transporters of S. typhimurium (Landmesser et al., 2002
; Liu & Ames, 1997
), only proteoliposomes with the ArtP subunits facing the medium would contribute to ATPase activity. As shown in Fig. 2
, proteoliposomes prepared in the absence of ArtJ and arginine exhibited only marginal ATPase activity; however, the activity was markedly stimulated by ArtJ. Moreover, loading the proteoliposomes with ArtJ and L-arginine further increased the initial rate of ATP hydrolysis by a factor of 1·6. Similar rates of substrate-dependent stimulation of ATPase activity were reported for the maltose (Landmesser et al., 2002
) and histidine (Liu et al., 1997
) transporters of S. typhimurium. Consistent with the growth conditions of G. stearothermophilus, ATPase activity was optimal at a temperature of 60 °C (not shown). Analysis of kinetic parameters revealed a maximal velocity of 0·71 µmol Pi min1 per mg protein (of total complex) and a Km (ATP) of 1·59 mM. Furthermore, the binding-protein-stimulated catalytic activity was sensitive to the specific inhibitor orthovanadate, thereby indicating functional coupling of ATP hydrolysis to substrate transport (Hunke et al., 1995
). Vanadate inhibition is caused by trapping of ADP in the binding pocket after hydrolysis of the
-phosphate of ATP (Sharma & Davidson, 2000
). Half-maximal inhibition was observed at a concentration of 40 µM, which confirms data reported for other ABC importers (Schneider, 2003
) (data not shown).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 August 2004;
revised 17 November 2004;
accepted 22 November 2004.
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