Escherichia coli enzyme IIA^Ntr regulates the K⁺ transporter TrkA

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   Fig. 1.

   Specific interaction of TrkA with EIIA^Ntr. Ligand fishing to search for protein factor(s) interacting with EIIA^Ntr. Crude extract prepared from MG1655 grown in 500 ml of LB to A ₆₀₀ of 2.0 was mixed with 2 mg of either purified His–EIIA^Ntr (lane 1) or EIIA^Ntr (lane 2), and these mixtures were passed through metal-affinity chromatography columns containing 500 μl of TALON resin. After washing the columns with 20 mM Hepes buffer (pH 7.5) containing 200 mM NaCl three times (2 ml), proteins bound to the resin were eluted with the same buffer (500 μl) containing 200 mM imidazole, and the eluates were analyzed by SDS/PAGE followed by staining with Coomassie brilliant blue (Bio-Rad, Hercules, CA). In-gel digestion followed by MALDI-TOF analysis revealed that the protein band bound specifically to His–EIIA^Ntr corresponded to TrkA (marked with an arrow). Tefco (Tokyo, Japan) Wide Range protein standards were used as molecular mass markers (lane M).

   
   
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   Fig. 2.

   Analysis of the interaction between EIIA^Ntr and TrkA by surface plasmon resonance. (A) Specific interaction of TrkA with EIIA^Ntr. Purified EIIA^Ntr and EIIA^Glc were immobilized on the carboxymethylated dextran surface of a CM5 sensor chip. Based on the assumption that 1,000 resonance units (RUs) corresponds to a surface concentration of 1 ng/mm², the proteins EIIA^Glc and EIIA^Ntr were immobilized to surface concentrations of 1.5 and 2.0 ng/mm², respectively. TrkA (5 μg/ml) was allowed to flow over the EIIA^Ntr (sensorgram a) and EIIA^Glc (sensorgram b) surfaces for 12 min each. The dissociation constant K _d for the interaction between EIIA^Ntr and TrkA was determined by using BIAevaluation 2.1 software (BIAcore, Uppsala, Sweden) to be ≈8.4 × 10⁻⁷ M. (B) Phosphorylation state-dependent interaction between TrkA and EIIA^Ntr. TrkA (5 μg/ml) was allowed to flow over the EIIA^Ntr surface for 5 min in each sensorgram. The phosphorylated and dephosphorylated EIIA^Ntr surfaces were generated by reversible phosphoryl transfer reactions between NPr and EIIA^Ntr, as described in Materials and Methods. Sensorgram a shows TrkA binding to the immobilized EIIA^Ntr surface without any treatment. In sensorgram b, TrkA was injected after the immobilized EIIA^Ntr surface had been phosphorylated by exposing it to a mixture of EI^Ntr and NPr in the presence of phosphoenolpyruvate (PEP), then flushing with running buffer to remove PEP and other PTS proteins. In sensorgram c, dephosphorylated NPr was allowed to flow over the phosphorylated EIIA^Ntr surface generated in sensorgram b to dephosphorylate the surface before TrkA was injected.

   
   
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   Fig. 3.

   The toxic effect of the Ala–Leu dipeptide on the ptsN mutant strain is neutralized by disruption of the trkA gene. Cells grown overnight in LB were harvested, washed, and suspended in M9 minimal medium containing 0.5% glucose and 0.5 mM Ala–Leu dipeptide. MG1655, Open circles; ptsP mutant CR101, open squares; ptsO mutant CR201, filled circles; ptsN mutant CR301, filled squares; and ptsN trkA double mutant CR302, filled triangles. Strains CR102 (the ptsP trkA double mutant) and CR202 (the ptsO trkA double mutant) showed growth curves similar to that of CR302.

   
   
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   Fig. 4.

   EIIA^Ntr-mediated change in AHAS expression depends on TrkA. (A) AHAS activity assays. Cells of strains MG1655, CR301 (ptsN), CR302 (ptsN TrkA), and EB101 (trkA) grown in LB were harvested, washed, and resuspended in K₀ minimal medium containing 0.5% glucose (filled bars) and K₂₀ minimal medium containing 0.5% glucose (open bars). Subsequently, cells were allowed to grow to midlogarithmic phase, and the specific activities of AHAS were measured as described in Materials and Methods. (B) Measurement of the ilvBN transcript by RT-PCR, which was carried out as described previously (15). (C) Primer extension analysis of the ilvBN transcript. RNA was prepared from the indicated cells grown to midlogarithmic phase in M9 minimal medium containing 0.5% glucose by using an RNeasy mini kit (Qiagen), and primer extension assays were carried out as described previously (5).

   
   
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   Fig. 5.

   Analysis of intracellular accumulation of K⁺. Strains MG1655, CR201, and CR301 grown in K₀ medium were harvested, washed, and resuspended in K₂₀ minimal medium containing 0.5% glucose. Cells were harvested by filtration at the indicated time points and processed for analysis of K⁺ content by employing inductively coupled plasma/optical emission spectrometry (ICP-OES) as described in Materials and Methods. MG1655, diamonds; CR201 (ptsO), triangles; CR301 (ptsN), squares; and CR301/pCR3(H73A), circles.

   
   
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   Fig. 6.

   Effect of K⁺ on AHAS activity. MG1655 cells were grown to midlogarithmic phase in M9 minimal medium containing 0.5% glucose at 37°C. The harvested cell pellet was washed and resuspended in 50 mM Tris·HCl buffer (pH 7.5) containing 20 mM FAD. This suspension was passed through a French pressure cell at 10,000 psi. The lysate was centrifuged at 100,000 × g for 90 min, and the supernatant was used as crude AHAS. The specific activity of AHAS was measured as previously described (15). To check the direct effect of K⁺ on AHAS activity, KCl was added to the reaction mixture to the indicated concentrations.