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

Abstract

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K⁺, cytoplasmic K⁺ in bacteria is required for diverse processes. Here, we show that enzyme IIA^Ntr (EIIA^Ntr) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K⁺ transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIA^Ntr was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIA^Ntr for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K⁺ level in culture media is lowered. We demonstrate that dephosphorylated EIIA^Ntr, but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K⁺. High cellular K⁺ levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIA^Glc regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIA^Ntr regulates the transport system for K⁺, which has global effects related to nitrogen metabolism.

The well defined phosphotransferase system (PTS) is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein (HPr), and some sugar-specific components collectively known as enzymes II (1). The carbohydrate PTS, especially for glucose uptake, occupies a central position in bacterial physiology as a result of the identification of multiple regulatory functions superimposed on the transport functions, such as regulation of chemotaxis by EI (2); regulation of glycogen breakdown by HPr (3, 4); regulation of the global repressor Mlc by the membrane-bound glucose transporter EIICB^Glc (5–7); and regulation of carbohydrate transport and metabolism (1, 8, 9), the metabolic flux between fermentation and respiration (10), and adenylyl cyclase activity (11) by EIIA^Glc. These regulatory functions of the carbohydrate PTS depend on the phosphorylation state of the involved components, which have been shown to increase in the absence and decrease in the presence of a PTS sugar substrate.

The nitrogen-metabolic PTS consists of enzyme I^Ntr (EI^Ntr, an EI paralog encoded by ptsP), NPr (an HPr paralog encoded by ptsO), and enzyme IIA^Ntr (EIIA^Ntr, an EIIA^Mtl paralog encoded by ptsN) (12–14). The genes encoding NPr and EIIA^Ntr are located on the same operon as rpoN, which led to the proposal that this pathway is involved in nitrogen metabolism. Because phosphoryl transfer to a specific substrate has not yet been demonstrated for the nitrogen PTS, it has been suggested that the role of this pathway is in regulation (12). In contrast to the extensive body of data dealing with regulation mechanisms associated with the carbohydrate PTS, substantially less is known about regulatory mechanisms connected with the nitrogen-metabolic PTS.

Recently, we reported that dephosphorylated EIIA^Ntr is required for derepression of the ilvBN operon, which encodes acetohydroxy acid synthase I (AHAS I), the enzyme catalyzing the first step common to the biosynthesis of the branched-chain amino acids (15). Because the derepression effect was only observed under specific growth conditions, we proposed that dephosphorylated EIIA^Ntr binds to some undefined regulator for that operon. Here, we used ligand fishing to isolate a protein factor interacting with EIIA^Ntr that regulates ilvBN expression and leucine sensitivity in Escherichia coli. The binding partner was identified as TrkA, a K⁺ transporter. We demonstrate that K⁺ serves as an important signal controlling both the transcription and activity of AHAS I.

Results

Specific Interaction Between the Dephosphorylated Form of EIIA^Ntr and the K⁺ Transporter TrkA.

We previously proposed that an additional unknown factor was involved in the requirement of dephosphorylated EIIA^Ntr for the derepression of the ilvBN operon. To search for a factor that interacts with dephosphorylated EIIA^Ntr, we used ligand fishing. A crude extract from E. coli MG1655 was mixed with EIIA^Ntr or a His⁶-tagged form of EIIA^Ntr (His–EIIA^Ntr) and subjected to pull-down assays by using a metal-affinity resin. We found a protein band (apparent molecular mass of ≈50 kDa) specifically eluting in a fraction containing His–EIIA^Ntr (Fig. 1). Peptide mapping of the protein band indicated that it corresponds to the K⁺ transporter TrkA, a cytoplasmic component of the low-affinity K⁺ transport system, Trk (16).

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 A₆₀₀ 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).

To explore whether formation of a TrkA–EIIA^Ntr complex occurs in vivo, TrkA with or without N-terminal His⁶ residues was expressed in cells lacking the chromosomal trkA gene. Crude extracts prepared from these cells were subjected to a pull-down assay by using an affinity resin. His⁶-tagged TrkA (His–TrkA) was bound to a Co²⁺-affinity column [see supporting information (SI) Fig. 7A, lane 4], and proteins eluted with 200 mM imidazole were analyzed by SDS/PAGE. EIIA^Ntr was coeluted with His–TrkA bound to the beads, whereas no EIIA^Ntr was recovered from the elution fraction when crude extract prepared from cells expressing unmodified TrkA was used (SI Fig. 7B). The tight in vivo interaction between EIIA^Ntr and TrkA was also confirmed by using the plasmid pCRduet coexpressing His–EIIA^Ntr and TrkA. When the crude extract prepared from cells overexpressing the two proteins was loaded onto a TALON metal-affinity column, TrkA copurified with His–EIIA^Ntr (see SI Fig. 8). These results support the contention that the tight interaction between TrkA and EIIA^Ntr also occurs in vivo.

To measure the binding constant between TrkA and EIIA^Ntr, the technique of surface plasmon resonance was used (4). EIIA^Ntr was immobilized on a CM5 sensor chip, and TrkA was allowed to flow over the surface. Because the similarity of the sequence identity between EIIA^Glc and EIIA^Ntr is only ≈13%, immobilized EIIA^Glc was used as a control. When purified TrkA was exposed to immobilized EIIA^Glc, no interaction was detected (Fig. 2A, sensorgram b). In contrast, TrkA bound to immobilized EIIA^Ntr (Fig. 2A, sensorgram a). The dissociation constant (K_d) for the interaction between EIIA^Ntr and TrkA was determined to be ≈8.4 × 10⁻⁷ M.

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.

Because the interaction with target proteins and regulatory functions of the PTS depend on the phosphorylation state of the involved proteins (5), the effect of phosphorylation of EIIA^Ntr on its affinity for TrkA was checked (Fig. 2B). The phosphorylated and dephosphorylated EIIA^Ntr surfaces were generated by reversible phosphoryl transfer reactions between NPr and EIIA^Ntr. When purified TrkA was exposed to immobilized EIIA^Ntr, a high-affinity interaction was detected (Fig. 2B, sensorgram a). After immobilized EIIA^Ntr was phosphorylated by allowing a mixture of EI^Ntr and NPr in the presence of phosphoenolpyruvate (PEP) to flow over the surface and subsequently washing with running buffer, an interaction with TrkA was hardly detectable (Fig. 2B, sensorgram b). The same EIIA^Ntr surface recovered TrkA binding activity after dephosphorylation when we allowed dephosphorylated NPr to flow through the cell and then flushed it with running buffer (Fig. 2B, sensorgram c). These results provide direct evidence for a phosphorylation state-dependent interaction between TrkA and EIIA^Ntr.

Effect of K⁺ Concentration and TrkA on Growth and Sensitivity to Ala–Leu of a ptsN Mutant.

Based on the phosphorylation state-dependent interaction between the K⁺ transporter TrkA and EIIA^Ntr, we envisioned that EIIA^Ntr would regulate the activity of TrkA and therefore the intracellular level of K⁺. To verify this thesis, we tested the effect of medium K⁺ concentration on the specific growth rate of the exponentially growing cells of a ptsN mutant and WT. It was previously shown that growth of the ptsN mutant was significantly slower than WT in W salts medium (containing 93 mM K⁺ ion) (13), and the same growth phenotype was observed in M9 minimal medium (containing 22 mM K⁺), although growth of the ptsN mutant was comparable with that of WT in LB and M9 minimal medium supplemented with 1% casamino acids (data not shown). As shown in Table 1, the growth rate of the ptsN mutant was markedly slower than that of WT in media containing 20–120 mM K⁺. As the K⁺ concentration in M9 minimal medium decreased, however, the growth rate of the ptsN mutant increased. Interestingly, the ptsN mutant exhibited a growth rate similar to that of its parental strain, MG1655, in K₁ and K_0.1 media (n in K_n corresponds to the millimolar concentration of K⁺ in the M9 minimal medium). The same effects were observed in M9 minimal medium in which glucose was replaced with lactose or glycerol (data not shown).

Table 1.

Effect of K⁺ concentration on the growth rate of the ptsN mutant (CR301) in minimal medium

K+ concentration, mM	Growth rate, hr−1
	Wild type	ptsN mutant
0.1	0.78 ± 0.035	0.82 ± 0.055
1.0	0.79 ± 0.021	0.78 ± 0.042
5.0	0.81 ± 0.014	0.47 ± 0.037
10	0.82 ± 0.028	0.30 ± 0.071
20	0.83 ± 0.016	0.15 ± 0.011
40	0.82 ± 0.010	0.08 ± 0.033
60	0.82 ± 0.031	0.10 ± 0.004
120	0.81 ± 0.034	0.06 ± 0.012

Cells grown overnight in LB medium were harvested, washed with K₀ medium, and suspended in K_n medium as described in Materials and Methods. The growth rate at 37°C of the cells was monitored by measuring the OD₆₀₀.

We previously showed that a ptsN mutant was hypersensitive to growth inhibition by the dipeptide Ala–Leu in normal medium (14). From results showing that dephosphorylated EIIA^Ntr interacts with TrkA, it might be predicted that TrkA in ptsN mutant cells would exist in the free form, whereas TrkA would exist in a complex with EIIA^Ntr in ptsO and ptsP mutants because dephosphorylated EIIA^Ntr would accumulate in these mutants. If the nitrogen PTS is epistatic to TrkA in the regulation of sensitivity to LCPs, then the sensitivity of a trkA mutant to Ala–Leu should be independent of mutations in the nitrogen PTS. To test this idea, we constructed three double mutants by deleting the trkA gene in the ptsP, ptsO, and ptsN backgrounds. Although the ptsN mutant was extremely sensitive to LCPs, cells of the ptsN trkA double-mutant strain exhibited normal growth rates, comparable with that of MG1655 in the medium supplemented with 0.5 mM Ala–Leu dipeptide (Fig. 3). Noteworthily, the disruption of trkA in ptsP and ptsO strains neutralized resistance to Ala–Leu to the level seen in MG1655 (data not shown). These results implicate TrkA as the regulator operating downstream of EIIA^Ntr with respect to sensitivity to LCPs.

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.

EIIA^Ntr-Mediated Change in AHAS Expression Is Dependent on TrkA.

It was previously reported that the sensitivity of the ptsN mutant to LCPs is due to the low level of AHAS expression, leading to isoleucine pseudoauxotrophy by leucine (15). Thus, we examined the effect of a trkA deletion on AHAS activity (Fig. 4A). AHAS activity in the ptsN mutant cells was significantly increased in the K₀ medium compared with that in cells grown in the K₂₀ medium. Furthermore, the introduction of a trkA deletion mutation into the ptsN mutant strain restored AHAS activity to the level of the WT strain, indicating that expression of AHAS is regulated by TrkA. The AHAS level in the trkA mutant was insensitive to the level of K⁺ in the medium regardless of the presence of the ptsN mutation. This result is consistent with the data in Fig. 3 showing that the trkA mutant was insensitive to Ala–Leu regardless of the presence of the ptsN mutation, further indicating the dependence of sensitivity to LCPs on the intracellular AHAS level.

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).

It was previously shown that the dephosphorylated form of EIIA^Ntr is required for derepression of the ilvBN operon, although ilvIH expression is not affected by a ptsN deletion (15). Therefore, the effect of a trkA deletion as well as the effect of the K⁺ level in the medium on ilvBN expression were examined. First, the mRNA level of the ilvBN operon was measured by RT-PCR in MG1655, the ptsN mutant CR301 (15), and a ptsN trkA double mutant grown in M9 minimal medium (Fig. 4B). As reported previously (15), the ilvBN transcript level in the ptsN mutant grown in the typical M9 minimal medium containing 22 mM K⁺ was much lower than that in WT. However, ilvBN expression in the ptsN mutant grown in M9 minimal medium without K⁺ (K₀ medium) was derepressed to the level in WT cells. Significantly, the ilvBN transcript level in the ptsN trkA double mutant was similar to that in WT. To confirm the effect of the trkA deletion on the expression of ilvBN, we also carried out primer extension analysis of the ilvBN transcript. As shown in Fig. 4C, the ptsN deletion caused a significant decrease in ilvBN transcription. Although introduction of the ptsP mutation did not significantly change the ilvBN transcript level in the ptsN mutant, introduction of the trkA mutation recovered ilvBN expression in the ptsN mutant to the level in WT cells. The obvious conclusion is that PtsN-dependent ilvBN expression is mediated by TrkA.

Dephosphorylated EIIA^Ntr Inhibits TrkA-Mediated K⁺ Uptake.

Taken together, our results suggest that TrkA-dependent K⁺ homeostasis is altered in strains lacking the ptsN gene. It seems clear that low (or no) expression of AHAS in the ptsN mutant was due to the increased level of intracellular K⁺ resulting from the increased level of the free form of TrkA. Therefore, the rate of K⁺ accumulation was measured in strains lacking ptsN, ptsO, or a ptsN mutant harboring an expression vector for the H73A mutant form of EIIA^Ntr and compared with that in the WT cells. When cells were shifted from K₀ to K₂₀ medium, the K⁺ uptake rate of the ptsN mutant was higher, whereas that of the ptsO and ptsN cells harboring pCR3(H73A) was lower than those in MG1655 (Fig. 5). These data suggest that dephosphorylated EIIA^Ntr inhibits TrkA-mediated K⁺ uptake and indicate that an increase of the K⁺ level in the ptsN mutant associated with the presence of free TrkA negatively affects derepression of AHAS activity.

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.

Cytoplasmic K⁺ concentrations are maintained at relatively constant levels of 300–500 mM in E. coli cells growing in standard media by the activities of several K⁺ uptake and K⁺ efflux systems (17, 18). Thus, we tested whether AHAS activity is affected by changes in the concentration of K⁺. AHAS activity was inhibited by K⁺ when its concentration was >200 mM in a concentration-dependent manner (Fig. 6). Thus, we concluded that K⁺ inhibits both AHAS expression and activity.

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.

Discussion

The maintenance of ionic homeostasis in response to environmental changes is vital to all living cells. K is the major monovalent cellular cation in bacteria such as E. coli. Cytoplasmic K⁺ is required for processes as diverse as the activation of cytoplasmic enzymes, maintenance of cell turgor, homeostasis of cytoplasmic pH, and adaptation of cells to osmotic conditions (19, 20). K⁺ concentrations are maintained at relatively constant levels (300–500 mM) in E. coli by the activities of K⁺ uptake and efflux systems (17, 18). The high-affinity K⁺ uptake ATPase Kdp (K_m for K⁺ uptake of ≈2 μM) accumulates K⁺ at low external K⁺ concentrations, whereas its expression is repressed in K⁺-replete cells (21). At normal cell turgor, E. coli takes up K⁺ via two constitutively expressed systems, Trk (K_m for K⁺ uptake of 0.9–1.5 mM) and Kup (K_m for K⁺ uptake of ≈0.37 mM) (22). Trk has an ≈100-fold higher V_max than Kup has, implying that Trk constitutes the major K⁺ uptake system under normal growth conditions. Further, Trk, but not Kup, has relatively high specificity for K⁺ (19, 22).

It has been suggested that complex mechanisms regulate K⁺ homeostasis in response to a stimulus perceived by cells (19). Recently, it was reported that a protein kinase regulates the K⁺ transporter Akt1 in Arabidopsis thaliana (23). From the results presented here, it is evident that dephosphorylated EIIA^Ntr interacts with TrkA, resulting in the inhibition of Trk-mediated K⁺ uptake. The abnormally high level of K⁺ accumulated by the free form of TrkA in a ptsN mutant inhibits both AHAS expression and activity. This explains the extreme sensitivity of a ptsN mutant and the resistance of a trkA mutant to LCPs. It was previously reported that a mutation in the kefA gene encoding a K⁺ efflux system made E. coli K-12 cells sensitive to K⁺ in a synthetic medium (24, 25), indicating that accumulation of high intracellular K⁺ inhibits the growth of these cells. These results are consistent with our data showing that a ptsN mutant was also sensitive to high medium concentrations of K⁺ (Table 1). These data, in addition to those showing that sensitivity of the ptsN mutant to LCPs was neutralized in K⁺-depleted medium, confirm that the slower growth of the ptsN mutant compared with WT is due to higher intracellular K⁺ concentration.

We studied the relationship of intracellular K⁺ concentration to AHAS expression. It was recently reported that K salts have a direct inhibitory effect on RNA polymerase at ribosomal promoters (26). It was shown that K⁺ can act independently of macromolecular repressors or activators by virtue of its ability to directly inhibit RNA polymerase binding to ribosomal promoters. Therefore, K⁺ may have a similar inhibitory effect on RNA polymerase at the ilvBN promoter or K⁺ may act through an as yet unidentified regulatory component.

The biological importance of regulation of AHAS expression and activity by the EIIA^Ntr–TrkA complex is worthy of discussion. As pointed out previously (15), cells typically make more proteins involved in amino acid synthesis when they are exposed to an environment depleted of amino acids. Previous studies (27–32) led us to conclude that leucine, one of the branched-chain amino acids whose synthesis is catalyzed by the enzyme AHAS, acts as a second messenger that monitors the environmental amino acid level. Although the three branched-chain amino acids constitute the most abundant protein building blocks in E. coli (27), their synthetic pathways are more complex than those for other amino acids. The regulatory importance of the three branched-chain amino acids is also supported by the existence of three AHAS isozymes with different properties in E. coli. Further, leucine acts as a signaling molecule regulating the leucine-responsive regulatory protein, Lrp (28), a global regulator involved in modulating a variety of metabolic functions including the catabolism and anabolism of amino acids as well as pili synthesis (29). In general, Lrp-related operons functioning in biosynthetic pathways are stimulated by Lrp, whereas those involved in catabolic pathways are repressed. Furthermore, Lrp, via its potent regulation of gltBDF and control of intracellular glutamine concentration, plays a significant role in the regulation of nitrogen assimilation genes (30). The high degree of conservation of Lrp in prokaryotes suggests that it has an important role in all enterobacteria (31, 32), implying that leucine's role as a second messenger is widespread in bacteria.

It is interesting that the paralogous PTSs in E. coli exhibit paralogous regulatory activities: the nitrogen-metabolic PTS inhibits the K⁺ transporter TrkA by a physical interaction with dephosphorylated EIIA^Ntr, whereas dephosphorylated EIIA^Glc of the carbohydrate PTS inhibits several non-PTS permeases via physical interactions (1). Although the phosphorylation state of PTS proteins is known to be controlled by the availability of PTS sugars, the mechanism regulating the phosphorylation state of the nitrogen-metabolic PTS proteins is not understood. The presence of a NifA protein-like putative sensory domain in EI^Ntr suggests that this domain might sense the availability of a ligand that controls the phosphorylation state of the nitrogen-metabolic PTS. Although the search for such a ligand has not yet been successful, changes in nutritional environment seem to signal the nitrogen-metabolic PTS to regulate TrkA activity and intracellular K⁺ concentrations. K⁺-dependent changes in AHAS activity then regulate intracellular leucine levels. Subsequently, leucine regulates the expression of numerous genes involved in amino acid metabolism through Lrp. It remains to be established how many additional bacterial pathways are regulated by fluctuations in cellular K⁺ levels.

Materials and Methods

Bacterial Strains, Plasmids, and Culture Conditions.

The bacterial strains and plasmids used in this study are listed in SI Table 2 and their constructions are described in SI Materials and Methods. Bacteria were cultured as described previously (15, 33).

Purification of Overexpressed Proteins.

Purification of EIIA^Ntr and EIIA^Ntr (H73A) was accomplished as described previously (15). EI^Ntr, NPr, and TrkA were overexpressed in GI698 cells harboring pCR1, pCR2, and pEB1 and purified after procedures previously developed for overexpression and purification of EI (33), HPr (4), and FrsA (10), respectively. His–EIIA^Ntr was purified by using TALON metal-affinity resin (Clontech, Mountain View, CA) according to the manufacturer's instructions, and bound His–EIIA^Ntr was eluted with binding buffer containing 200 mM imidazole. The fractions containing His–EIIA^Ntr were pooled and concentrated in a Macrosep centrifugal concentrator (3K, Pall, East Hills, NY). To obtain homogeneous His–EIIA^Ntr (>98% pure) and to remove imidazole, the concentrated pool was chromatographed on a HiLoad 16/60 Superdex 75 prep grade column (Amersham Biosciences, Little Chalfont, England) equilibrated with buffer A [20 mM Hepes–KOH (pH 8.0), containing 200 mM NaCl].

Ligand Fishing to Search for Proteins Interacting with His⁶–Tagged EIIA^Ntr.

E. coli MG1655 cells grown overnight in 500 ml of LB were harvested and washed with and resuspended in 30 ml of buffer A in the presence of 100 μg/ml PMSF. Cells were disrupted by passing twice through a French pressure cell at 10,000 psi (1 psi = 6.89 kPa) and then by centrifuging at 100,000 × g for 60 min at 4°C. The supernatant was mixed with 2 mg of EIIA^Ntr or His–EIIA^Ntr, and this mixture was incubated with 500 μl of TALON metal-affinity resin. After incubation at room temperature for 10 min, the mixture was loaded onto a Poly-Prep chromatography column (8 × 40 mm) (Bio-Rad, Hercules, CA). The column was washed with 1 ml of buffer A three times, and the proteins bound to the column were eluted with buffer A containing 200 mM imidazole. Aliquots of the eluted protein sample (10 μl each) were analyzed by SDS/PAGE and then were stained with Coomassie brilliant blue R. The protein band specifically bound to His–EIIA^Ntr was excised from the gel, and in-gel digestion and peptide mapping of tryptic digests were carried out as described previously (34).

Surface Plasmon Resonance Spectroscopy.

Real-time interaction of TrkA with EIIA^Ntr or EIIA^Glc was monitored via surface plasmon resonance detection by using a BIAcore 3000 system (BIAcore, Uppsala, Sweden) with some modifications as described previously (4, 5, 10). EIIA^Ntr and EIIA^Glc were separately immobilized onto the carboxymethylated dextran surface of a CM5 sensor chip. EIIA^Glc and EIIA^Ntr (100 μl, 10 μg/ml) in coupling buffer (10 mM sodium acetate, pH 5.0) were allowed to flow over the sensor chip at 5 μl/min to couple the proteins to the matrix by a N-hydroxysuccinimide/1-ethyl-3′-(3-dimethylaminopropyl)carbodiimide reaction (80 μl of mix). Based on the assumption that 1,000 resonance units (RUs) corresponds to a surface concentration of 1 ng/mm², EIIA^Glc and EIIA^Ntr were immobilized to surface concentrations of 1.5 and 2.0 ng/mm², respectively. The standard running buffer was 10 mM Hepes (pH 7.2), 150 mM NaCl, 10 mM KCl, 1 mM MgCl₂, and 0.5 mM EDTA, and all reagents were introduced at a flow rate of 10 μl/min. The sensor surface was regenerated between assays by using the standard running buffer at a flow rate of 100 μl/min for 10 min to remove bound analytes.

To phosphorylate the immobilized EIIA^Ntr, a mixture (50 μl) of phosphoenolpyruvate (PEP) (0.5 mM), EI^Ntr (20 μg/ml), and NPr (10 μg/ml) in standard running buffer was allowed to flow into the flow cell for 10 min. To remove the phosphoryl group from the immobilized phosphorylated EIIA^Ntr, NPr (0.25 mg/ml in standard running buffer) was allowed to flow into the flow cell for 15 min. The sensor surface was regenerated between injections by running standard buffer at a flow rate of 100 μl/min for 10 min to remove bound analytes.

Effect of K⁺ Concentration on Growth and Sensitivity to Ala–Leu of the ptsN Mutant.

The influence of K⁺ concentration in the medium on growth rate was tested on the K_n minimal medium (pH 7.0), where n is the K⁺ concentration in micromolars (24, 35). K₁₂₀ medium consisted of 48 mM K₂HPO₄, 24 mM KH₂PO₄, 8 mM (NH₄)₂SO₄, 0.4 mM MgSO₄, 6 μM FeSO₄, 1 mM sodium citrate, 1 mg/ml thiamine hydrochloride, and 0.2% glucose. K₀ medium was similar, with equimolar amounts of Na salts replacing the potassium phosphate. K_n medium was prepared by mixing suitable proportions of K₀ and K₁₁₅. Fresh cells grown overnight in LB were harvested and washed with K₀ medium. After resuspension in K_n medium, specific growth rates of these cells at 37°C were determined by measuring the OD at 600 nm. Growth was also checked in the presence of 0.5 mM Ala–Leu dipeptide in K₀ medium where indicated.

Transcript Analysis.

RT-PCR was carried out as described previously (15). Primer extension reactions were carried out as described previously (16). Cells were grown to A₆₀₀ of 0.5 in M9 minimal medium containing 0.5% glucose, and total E. coli RNA was purified by using an RNeasy mini kit (Qiagen, Valencia, CA) and resuspended in sterile distilled water. Total cell RNA (30 μg) was mixed with 0.2 pmol of the γ-³²P-end-labeled primer ilvBPex, 5′-TTG CCA TGC TCC AGT CCT TTT CTT CTG GGC-3′. The mixture was heated to 60°C and then allowed to cool to room temperature over a period of 1 h. After annealing, 700 μM dNTPs, 10 mM MgCl₂, 5 mM DTT, 20 mM Tris·HCl (pH 8.3), and 100 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) were added to a final volume of 100 μl. After the mixture was incubated at 40°C for 70 min, 2 μl of 0.5 M EDTA was added to the reaction mixture, and the mixture then was incubated at 37°C for 30 min. DNA was precipitated and resolved on 5% polyacrylamide gel with 8 M urea and visualized by autoradiography.

Measurement of Intracellular K⁺ Concentration.

Intracellular K⁺ concentration was measured according to the procedure of McLaggan et al. (24) with some modifications. Cells cultured in LB were harvested by centrifugation, washed, and incubated at 37°C in K₀ medium. After incubation for 1 h, KCl was added to 20 mM, and the accumulation of K⁺ was determined. Samples (10 ml each) taken at the indicated time points were rapidly filtered on 47-mm membrane filters [pore size of 0.8 μm; Whatman (Brentford, U.K.) polycarbonate]. The samples were washed dropwise with 1–2 ml of K₀ medium (37°C) made slightly hypertonic by the addition of 50 mM NaCl. After the filters were dried, 10 ml of distilled water was added, and the mixtures were heated at 90°C for 10 min. The released K⁺ was measured by using an inductively coupled plasma/optical emission spectrometer (ICP-OES) (Shimadzu, Kyoto, Japan).

Abbreviations

PTS

phosphotransferase system

AHAS

acetohydroxy acid synthase

LCPs

leucine-containing peptides

PEP

phosphoenolpyruvate.