File S1 - The Sodium/Proline Transporter PutP of Helicobacter pylori

Table S1, Figures S1-S7. Table S1. Oligonucleotides. Figure S1. Alignment of the amino acid sequences of PutP of E. coli and H. pylori and SGLT of V. parahaemolyticus (vSGLT). The alignment was performed with the complete amino acid sequences of the transporters using CLUSTAL OMEGA followed by manual adjustment. Figure S2. Prediction of transmembrane helices in PutP of H. pylori. The analysis was performed with the TMHMM Server 2.0. Figure S3. Effect of the deletion of put genes on growth of H. pylori. Cells were grown in Brucella broth under microaerophilic conditions as described in Experimental Procedures. The optical density was determined at 600 nm at given time points. Average values and standard deviations were calculated from three parallel measurements. The entire experiment was independently repeated four times yielding similar results. Figure S4. Kinetics of 14C-L-proline uptake into H. pylori. Cells were grown in Brucella broth under microaerophilic conditions as described in Experimental Procedures. For the transport assay, 200 µl aliquots of a cell suspension (OD600=0.8 in 100 mM Tris/MES, pH 7.0/150 mM KCl) were prepared per time point. Initial rates of 14C-L-proline uptake were determined (A) at 14C-L-proline concentrations varying from 0.5 µM to 250 µM in the presence of 50 mM NaCl, and (B) at a constant 14C-L-proline concentration of 10 µM and NaCl added at concentrations varying from 0.07 mM to 250 mM using the rapid filtration assay. Data points represent the mean of duplicate determinations of a representative experiment. Three repeats of the experiment with independently grown and treated cells yielded similar km(Pro) and ko.5(Na+) values with maximum activities varying by a factor of up to three between the individual experiments. Figure S5. Comparison of the amounts of EcPutP and HpPutP in membranes of E. coli WG170. Expression of EcputP (Ec) and HpputP (Hp) was achieved form the promoters lac (plasmids pTHpputPF6H, pTEcputPF6H) and trc (plasmids pRHpputPF6H, pREcputPF6H). Plasmids pT7-5 and pTrc99a served as negative controls (nc). Relative amounts of the transporters were estimated by Western-Blot analysis with HRP-linked anti-FLAG IgG directed against the FLAG epitope at the C termini of EcPutP and HpPutP. Figure S6. Purification and reconstitution of HpPutP. Cells of E. coli WG170 transformed with plasmid pRHpputP6H were grown and membranes were prepared as described in Experimental Procedures. The protein (5 mg ml-1 total membrane protein) was solubilized with 1.5 % n-dodecyl-ß-D-maltopyranoside (DDM) and purified by Ni2+-NTA affinity chromatography. Out of 45 mg total membrane protein applied to 1 ml Ni2+-NTA agarose about 1 mg of HpPutP with a purity of about 95 % was obtained. (A) SDS-PAGE and Coomassie stain, (B) Western Blot of individual steps of the purification procedure. (C) Scheme of the reconstitution procedure. Liposomes were preformed from an E. coli polar lipid extract, detergent destabilized, incubated with purified HpPutP at a lipid to protein ratio of 100 to 1 (w/w), and proteoliposomes were formed by stepwise removal of the detergent with Bio-Beads SM2. Figure S7. Dixon plot analysis of the inhibition of 14C-L-proline uptake by (A) 3,4-dehydro-D,L-proline (DHP) and (B) L-azetidine-2-carboxylic acid (AZC). Uptake of 14C-L-proline (26 Ci mol-1) into E. coli WG170 harboring HpPutP was assayed in the presence of 50 mM NaCl and 20 mM D-lactate (Na+-salt) at the indicated inhibitor concentrations at 25°C. (PDF)