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Journal of Bacteriology, January 2004, p. 343-350, Vol. 186, No. 2
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.2.343-350.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of the LIV-I/LS System as the Third Phenylalanine Transporter in Escherichia coli K-12

Takashi Koyanagi, Takane Katayama, Hideyuki Suzuki, and Hidehiko Kumagai*

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

Received 7 October 2003/ Accepted 21 October 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain ({Delta}aroP {Delta}pheP {Delta}mtr {Delta}tna {Delta}tyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The Km values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 µM, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
It has been reported that Escherichia coli has five distinct transport systems (AroP, Mtr, PheP, TnaB, and TyrP) for the accumulation of aromatic amino acids (36). A general amino acid permease, encoded by the aroP gene, transports three aromatic amino acids with high affinity (8, 11, 21, 36). The closely related PheP protein transports phenylalanine in preference to tyrosine but does not exhibit tryptophan uptake activity (10, 34-36). Mtr and TyrP are specific for tryptophan and tyrosine, respectively (19, 36, 42, 51, 52), and TnaB is a low-affinity, tryptophan-specific transporter encoded in the tryptophanase operon together with the tnaA gene (13, 36, 43).

In a previous study, we cloned the tyrosine transporter tutB gene of Erwinia herbicola and used E. coli cells to determine the properties of its product (23). In the course of that study, we found that the aromatic amino acid transporter-deficient E. coli strain TK1135 ({Delta}aroP {Delta}pheP mtr24 {Delta}tna {Delta}tyrP) (23) has the ability to accumulate phenylalanine in an energy-dependent manner, although the initial rate of uptake, as well as the steady-state level, was quite low. This finding prompted us to examine the basis for this activity and whether this transport activity is physiologically important in E. coli. Here, we present evidence indicating that a branched-chain amino acid transporter, the LIV-I/LS system (1-3, 18, 24, 28, 29, 32, 37, 38, 45, 50), acts as the third phenylalanine transporter, plays a significant role in the accumulation of phenylalanine, and has a broader substrate specificity than previously reported.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Bacterial strains and plasmids. The bacterial strains used in this study are derivatives of E. coli K-12. The strains and plasmids are listed in Table 1 with their characteristics. The aroP, pheP, tna, and tyrP genes were disrupted as described previously (23), and the mtr gene was disrupted, using mtr-1 and mtr-2 (Table 1) as primers, by the method described by Datsenko and Wanner (12). Disruption of the brnQ gene was carried out as follows. The brnQ gene was amplified by PCR using KOD polymerase (Toyobo, Japan) with the genomic DNA of MG1655 as the template and brnQ-F and brnQ-R (Table 1) as the primer pair, and the amplified DNA fragment was ligated with the 3.5-kb NsiI (blunt-ended)-NruI fragment of pACYC177 (6). The internal region of the brnQ gene was then removed by EcoRV-PvuII digestion and replaced with the Flp recognition target (FRT)-flanked kanamycin resistance gene (FRT-kan+-FRT), which was amplified by PCR using pKD13 (12) as the template and pKD13-1 and pKD13-4 (Table 1) as the primer pair. The resulting {Delta}brnQ::(FRT-kan+-FRT) gene was introduced into strain MG1655 harboring pKD46 (12) by electroporation and allowed to integrate into the chromosome through a double-crossover event. Elimination of the kan gene from the integrated locus was carried out as described previously (12), with the aid of plasmid pCP20 carrying the Flp recombinase gene (7). Disruption of the livHMGF region and the livJ-yhhK-livKHMGF gene cluster was performed similarly. In these cases, primer pair livH-F and livF-R and primer pair liv-F1 and liv-R1 (Table 1) were used for amplification of the livHMGF and livJ-yhhK-livKHMGF genes, respectively. The internal 2.9-kb BglII-PvuII region in the livHMGF cluster and the 6.4-kb PvuII region within the livJ-yhhK-livKHMGF cluster were deleted and replaced with the FRT-kan+-FRT gene. Integration into the chromosome and subsequent elimination of the kan+ gene were carried out as described above.


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  		TABLE 1. Strains, plasmids, and oligonuclotides used in this work

 
After confirmation of the correct recombination event by Southern hybridization analysis (41) and/or genomic PCR analysis with primers designed to anneal external regions that had been used for the homologous recombination event, the disrupted gene was transferred to other strains by P1 transduction (26).

Media and chemicals. Luria-Bertani (LB) (26) broth was routinely used for the cultivation of E. coli strains. M63-glucose (26) was used as the minimal medium, and, when necessary, phenylalanine and pantothenate were added as growth requirements to final concentrations of 10 µM to 1 mM and 5 µg/ml, respectively. Ampicillin, tetracycline, and kanamycin were used at final concentrations of 100, 15, and 30 µg/ml for LB medium and 50, 7.5, and 15 µg/ml for the minimal medium, respectively. For the disk inhibition assay, disks were impregnated with 1 mM concentrations of various amino acids and then put onto the plates. L-(U-14C)-isoleucine (314 mCi/mmol, 0.05 mCi/ml), L-(U-14C)-leucine (306 mCi/mmol, 0.05 mCi/ml), L-(U-14C)-valine (256 mCi/mmol, 0.05 mCi/ml), and L-(U-14C)-tyrosine (434 mCi/mmol, 0.05 mCi/ml) were purchased from Amersham Pharmacia Biotech. L-(U-14C)-phenylalanine (496 mCi/mmol, 0.1 mCi/ml) and L-(side chain-3-14C)-tryptophan (58.1 mCi/mmol, 0.02 mCi/ml) were from Perkin-Elmer Life Sciences Inc. The chemicals were all obtained commercially and not purified further.

Genetic techniques. Standard genetic techniques were used essentially as described by Sambrook and Russell (41). The method used for generalized transduction involving the P1 phage was that described by Miller (26).

Cloning of the liv gene cluster. The chromosomal locus including the liv gene cluster consists of the livJ, yhhK, livK, livH, livM, livG, and livF genes in that order. While the livJ and livK genes encode periplasmic binding proteins, the livH, livM, livG, and livF genes specify membrane channel components (1, 45). The function of the yhhK gene has not been clarified yet. The DNA fragment containing the livJ-yhhK-livK region was amplified by high-fidelity PCR using KOD polymerase (Toyobo, Japan) with the genomic DNA of MG1655 as the template and liv-F1 and liv-R2 (Table 1) as the primer pair. To clone the livJ gene, the amplified fragment was digested with AatI to remove the yhhK and livK genes and then inserted into the SalI (blunt-ended) site of pYG249. The livK gene was recovered by BglII digestion of the amplified fragment, blunt ended, and then inserted into the SalI (blunt-ended) site of pYG249. The amplified livK and livJ genes were entirely sequenced to ensure that no misincorporation of nucleotides had occurred during the PCR amplification.

The genes for the livHMGF cluster were cloned as follows. The DNA fragment containing the livJ-yhhK-livKHMGF gene cluster was amplified by high-fidelity PCR with the genomic DNA of MG1655 as the template and liv-F1 and liv-R1 (Table 1) as the primer pair. After insertion of the fragment into the PvuII site of pMW118 (Nippon Gene, Tokyo, Japan), the 2.3-kb EcoRV fragment containing the livJ-yhhK-livK genes was removed and the remaining large fragment carrying the livHMGF genes was circularized by self-ligation. Although the livKHMGF genes constitute an operon and are usually transcribed in one unit, it has been shown that a weak internal promoter present just upstream of the livH gene can direct synthesis of the downstream genes (1). Sequence analysis of the amplified fragment revealed a two-base discordance compared to data reported by Blattner et al. (4) at a locus downstream of the stop codon of the livF gene, which would have no substantial effect on the properties of the LIV-I/LS system. The resulting plasmid, pYG218, was introduced into strain YG201 ({Delta}aroP {Delta}brnQ {Delta}livHMGF {Delta}pheP) and examined for the ability to complement the chromosomal livHMGF lesion with respect to phenylalanine transport.

Transport assays. Transport assays were performed as described previously (23, 51), with slight modifications as follows. Cells grown in minimal medium were harvested at mid-exponential phase and then washed twice with M63-glucose containing 60 µg of chloramphenicol/ml to stop protein synthesis. The assay was initiated by adding the cell suspension to the reaction mixture containing various concentrations of labeled substrates in the presence or absence of cold competitive inhibitors. The rate of nonspecific diffusion was determined using energy-starved cells that had been prepared by incubating cells in the presence of 100 µM carbonylcyanide-m-chlorophenylhydrazone (CCCP) for 30 min prior to starting the assay. The uptake of substrates was expressed as picomoles per milligram of dry cells as a function of time.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
It has so far been considered that in E. coli, the active transport of aromatic amino acids across the inner membrane is mediated by five distinct permeases, AroP, Mtr, PheP, TnaB, and TyrP (8, 10, 11, 13, 19, 21, 34-36, 42, 43, 51, 52) and that among them, the AroP and PheP systems are responsible for the accumulation of phenylalanine in cells. However, as described above, in the course of studying tyrosine transporter TutB of E. herbicola through the use of E. coli cells (23), a low level of accumulation of phenylalanine was observed in the aromatic amino acid transporter-deficient strain TK1135 ({Delta}aroP {Delta}pheP mtr24 {Delta}tna {Delta}tyrP) (data not shown). At first we speculated that this activity might be due to altered specificity of the mutant Mtr protein, i.e., Mtr24 (20), although the nature of the mtr24 allele has not been elucidated. This possibility, however, was ruled out by the observation that an E. coli strain, TK1170 ({Delta}aroP {Delta}pheP {Delta}mtr {Delta}tna {Delta}tyrP), accumulated as much phenylalanine (Fig. 1B) as strain TK1135 in an energy-dependent manner. Even though the initial rate of uptake and the steady-state level of phenylalanine in cells were not so high compared to those with known phenylalanine transport systems, AroP and PheP, reported previously (5, 49) (Fig. 2), this activity seems to be important for cells to accumulate phenylalanine because a phenylalanine-auxotrophic (Phe-) strain could be obtained by transducing TK1170 ({Delta}aroP {Delta}pheP {Delta}mtr {Delta}tna {Delta}tyrP) with a P1 phage lysate prepared from strain NK6024 (pheA18::Tn10) (PheA, chorismate mutase-prephenate dehydratase) and subsequent selection with Tn10 as a marker. These findings suggested that E. coli might have at least one additional phenylalanine transporter.



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  		FIG. 1. Inhibition of phenylalanine uptake by branched-chain amino acids. (A) Growth inhibition of phenylalanine-auxotrophic (Phe-) E. coli strain TK1173 ({Delta}aroP {Delta}mtr {Delta}pheP {Delta}tna {Delta}tyrP pheA18::Tn10) in the presence of branched-chain amino acids, as observed on disk assaying. Cells were grown in LB medium, washed twice with M63 minimal buffer, mixed with the top agar, and then overlaid on M63 minimal solid medium containing 100 µM phenylalanine. Disks were impregnated with 1 mM concentrations of various amino acids (indicated by a one-letter code). (B) Phenylalanine uptake activity of E. coli strain TK1170 ({Delta}aroP {Delta}mtr {Delta}pheP {Delta}tna {Delta}tyrP). L-Phenylalanine was added to cell suspensions to a final concentration of 50 µM in either the absence ({square}) or presence of 5 µM glutamate ({circ}), leucine ({blacksquare}), or valine (•). Samples were withdrawn at the indicated times. The experiments were repeated three times with essentially the same results; the data for a representative experiment are shown.

 


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  		FIG. 2. The LIV-I/LS system as the third phenylalanine transporter in E. coli. L-Phenylalanine uptake was measured in various E. coli cells, including YG74 ({Delta}aroP {Delta}livHMGF {Delta}pheP) (BrnQ) ({blacktriangleup}), YG106 ({Delta}aroP {Delta}brnQ {Delta}pheP) (LIV-I/LS) ({square}), YG108 ({Delta}aroP {Delta}brnQ {Delta}livHMGF) (PheP) (•), YG109 ({Delta}brnQ {Delta}livHMGF {Delta}pheP) (AroP) ({triangleup}), and YG201 ({Delta}aroP {Delta}brnQ {Delta}livHMGF {Delta}pheP) ({circ}) cells, and compared to that in wild-type strain MG1655 ({blacksquare}). Cell suspensions were incubated in the presence of 1 µM L-(U-14C)-phenylalanine, and samples were withdrawn at the times indicated. The experiments were repeated three times with essentially the same results; the data for a representative experiment are shown.

 
Inhibition of phenylalanine uptake by branched-chain amino acids. The question is what system is involved in this transport: a protein encoded by an undefined open reading frame (function unknown ORF) or a defined small molecule transporter with broad substrate specificity? We found by using a disk inhibition assay (Fig. 1A) that the growth of the aromatic transporter-negative Phe- strain TK1173 ({Delta}aroP {Delta}pheP {Delta}mtr {Delta}tna {Delta}tyrP pheA18::Tn10) in minimal medium supplemented with phenylalanine was severely inhibited in the presence of isoleucine and leucine. In this assay, valine and serine were omitted since both cause a serious growth defect by blocking the synthesis of intermediates required for the synthesis of other amino acids (17, 25, 44). While large clear zones of inhibition were observed around the disks impregnated with isoleucine and leucine, a small inhibition zone was also observed around the disk impregnated with threonine (Fig. 1A), which is discussed later. No significant inhibition zones appeared around the other 15 amino acids tested. The results in Fig. 1A suggested that phenylalanine might be accumulated in cells through a branched-chain amino acid transport system. This notion was further supported by a transport assay with L-(U-14C)-phenylalanine. Whereas the presence of glutamate (Fig. 1B) in the assay mixture did not affect phenylalanine uptake, the addition of valine and leucine decreased the phenylalanine uptake activity even with a low concentration (5 µM valine and leucine each versus 50 µM phenylalanine). These results strongly suggested that the active transport of phenylalanine into E. coli cells with the {Delta}aroP {Delta}pheP {Delta}mtr {Delta}tna {Delta}tyrP background could be dependent on a system that transports branched-chain amino acids.

Identification of the LIV-I/LS system as the third phenylalanine transporter in E. coli. Branched-chain amino acids are transported into E. coli cells by an osmotic-shock-sensitive system designated LIV-I/LS (1-3, 18, 24, 28, 29, 32, 37, 38, 45, 50) and by an osmotic-shock-resistant system, BrnQ (15, 16, 31, 45, 53, 54), formerly called LIV-II (3, 31, 37, 38, 45, 50). Whereas transport by the BrnQ system is mediated by a single membrane protein (38, 45, 50), uptake by the LIV-I/LS system depends on two substrate-binding proteins (BP), LIV-BP and LS-BP, located in the periplasm (2, 14, 24, 33, 38, 45, 50). Previous studies involving purified BPs showed that LIV-BP, encoded by the livJ gene, binds isoleucine, leucine, and valine with Kd values of 10-6 to 10-7 M and threonine, serine, and alanine with lower affinity and that LS-BP, encoded by the livK gene, binds leucine with a Kd value of approximately 10-6 M (24). To enable the ATP-hydrolysis-coupled transport of their substrates into the cytoplasm, LIV-BP and LS-BP interact with the common inner-membrane components LivHMGF, which constitute the LIV-I and LS systems, respectively (1, 28, 29, 45, 50). These six liv genes are clustered at 77 min on the chromosome (45) and divided into two transcription units, one for livJ and the other for livKHMGF (1, 18, 45). In the region between livJ and livK there is the yhhK gene; the deletion of this region results in pantothenate auxotrophy (1).

To determine whether BrnQ or LIV-I/LS carries out the uptake of phenylalanine, a series of E. coli strains expressing individual transport systems was constructed and assayed for transport: AroP-expressing strain YG109 ({Delta}brnQ {Delta}livHMGF {Delta}pheP), BrnQ-expressing strain YG74 ({Delta}aroP {Delta}livHMGF {Delta}pheP), LIV-I/LS-expressing strain YG106 ({Delta}aroP {Delta}brnQ {Delta}pheP), and PheP-expressing strain YG108 ({Delta}aroP {Delta}brnQ {Delta}livHMGF). The transport activity was measured in the presence of 1 µM labeled phenylalanine and compared to that of wild-type strain MG1655 and strain YG201 lacking portions of the aroP, brnQ, livHMGF, and pheP genes.

As shown in Fig. 2, neither BrnQ-expressing strain YG74 ({Delta}aroP {Delta}livHMGF {Delta}pheP) nor strain YG201 ({Delta}aroP {Delta}brnQ {Delta}livHMGF {Delta}pheP) could accumulate phenylalanine. A sodium gradient made by adding NaCl (final concentration, 1 mM) to the assay mixture did not have any effect on the uptake activity of these strains. On the other hand, LIV-I/LS-expressing strain YG106 ({Delta}aroP {Delta}brnQ {Delta}pheP) was able to accumulate phenylalanine, demonstrating the involvement of the LIV-I/LS system, but not BrnQ, in phenylalanine transport, although the initial rate and the steady-state level were considerably lower than those in the strains expressing AroP and PheP. It seemed likely that the small inhibition halo observed around the disk impregnated with threonine shown in Fig. 1A reflected the substrate preference of LIV-BP (24). Despite the low phenylalanine transport activity, the LIV-I/LS system alone could support the growth of Phe- strain YG210 ({Delta}aroP {Delta}brnQ {Delta}pheP pheA18::Tn10) in minimal medium supplemented with 10 µM phenylalanine, indicating the participation of the LIV-I/LS system in the accumulation of phenylalanine. The Km value for phenylalanine in the LIV-I/LS system was determined to be 30 µM, which is considerably higher than those for AroP (0.4 µM) and PheP (2 µM) (36).

AroP-expressing strain YG109 ({Delta}brnQ {Delta}livHMGF {Delta}pheP) exhibited the highest uptake activity, and its activity was essentially equal to that of wild-type strain MG1655, suggesting that the AroP protein ordinarily acts as the major phenylalanine transport system in wild-type cells. As for PheP-expressing strain YG108 ({Delta}aroP {Delta}brnQ {Delta}livHMGF), more than 40 pmol of phenylalanine/mg (dry weight of cells) was accumulated in the cells, which was comparable to the steady-state level in the case of the AroP system, although the initial rate of uptake was significantly lower than that for AroP.

Next, we tested which binding protein, LIV-BP or LS-BP, participates in the transport of phenylalanine. For this end, LIV-BP and LS-BP were expressed in E. coli cells individually in the presence of membrane machinery components LivHMGF. Strain YG228 [{Delta}aroP {Delta}brnQ {Delta}(livJ-yhhK-livKHMGF) {Delta}pheP] was transformed with two compatible plasmids; one was a pSC101-derived vector carrying the genes for membrane components LivHMGF (pYG218), and the other was a Mini-F-derived plasmid carrying either the livJ gene (LIV-I) (pYG237; Fig. 3A) or the livK gene (LS) (pYG239; Fig. 3B). These strains were used for uptake assay in the presence of 10 to 300 µM phenylalanine (Fig. 3A and B). The results clearly show that both BPs are capable of effecting transport of the substrate. The amounts of phenylalanine accumulated in the cells significantly differed between them, but we cannot comment about this difference because the organization of the liv genes on plasmids was different from that on the chromosome. In the absence of BP, no accumulation was observed in the cells (Fig. 3A and B). Considering that the disruption of livHMGF, the genes encoding the membrane components, completely abolished phenylalanine transport (Fig. 2), it can be concluded that both BPs interact only with LivHMGF.



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  		FIG. 3. Uptake studies of various amino acids with E. coli cells expressing the LIV-I (A) and LS (B) systems. (A) Strain YG228 [{Delta}aroP {Delta}brnQ {Delta}(livJ-yhhK-livKHMGF) {Delta}pheP] carrying pYG218 (pSC101 replicon bla+ livH+M+G+F+) (membrane components) was transformed with either pYG237 (Mini-F replicon kan+ livJ+) (LIV-I; open symbols) or pYG249 (Mini-F replicon kan+) (control; filled symbols). For the tyrosine transport assay, YG256 [{Delta}aroP {Delta}brnQ {Delta}(livJ-yhhK-livKHMGF) {Delta}pheP {Delta}tyrP] was used instead of YG228. Cell suspensions were incubated in the presence of 10 to 300 µM L-phenylalanine ({square}, {blacksquare}) and 25 to 300 µM L-tyrosine ({triangledown}, {blacktriangledown}). (B) Strain YG228 carrying pYG218 was transformed with either pYG239 (Mini-F replicon kan+ livK+) (LS; open symbols) or pYG249 (control; filled symbols). Cell suspensions were incubated in the presence of 10 to 300 µM L-phenylalanine ({square}, {blacksquare}), 0.4 to 70 µM L-leucine ({diamond}, {diamondsuit}), L-isoleucine ({circ}, •), or L-valine ({triangleup}, {blacktriangleup}). All experiments were repeated three times with essentially the same results; the data for a representative experiment are shown.

 
Recently, using NMR with fluorine-labeled LS-BP, Salopek-Sondi and Luck revealed that LS-BP binds phenylalanine in addition to leucine and that the phenylalanine-binding ability is specific for LS-BP but not for LIV-BP (39). Our transport studies corroborated the ability of LS-BP to bind phenylalanine but contradicted the results obtained for LIV-BP.

Taken together, these results led us to the conclusion that in E. coli, there are three phenylalanine uptake systems, AroP, PheP, and LIV-I/LS, all of which may allow phenylalanine accumulation.

Kinetic studies of the LIV-I and LS systems. To further characterize the LIV-I and LS systems, kinetic constants for both systems were determined by monitoring phenylalanine uptake in the absence or presence of probable competitive inhibitors, branched amino acids. The Km values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 µM, respectively, by double-reciprocal plotting of the data in Fig. 3 (Table 2). In inhibition assays, as expected from the substrate specificity of LIV-BP, phenylalanine uptake by the LIV-I system was found to be decreased in a concentration-dependent manner upon the addition of isoleucine, leucine, and valine (data not shown), the Ki values for them having been determined to be 2.3, 1.7, and 1.5 µM, respectively (Table 2). These Ki values were comparable to the respective Km values determined by means of transport assays with LIV-I-expressing cells incubated in the presence of 0.4 to 70 µM labeled branched-chain amino acids (Table 2). The Ki values for valine and phenylalanine inhibition of leucine uptake were also determined by incubating cells under conditions of 0.4 to 20 µM labeled leucine in the presence of cold valine (0.5 to 20 µM) and phenylalanine (15 to 50 µM). The values obtained (Ki = 1.4 µM for valine and 30 µM for phenylalanine) were in good agreement with the Km values (2.4 µM for valine and 19 µM for phenylalanine) (Table 2).


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  		TABLE 2. Kinetic constants (Km and Ki) for various substrates in the LIV-I and LS systems as determined by in vivo uptake assays

 
The finding that phenylalanine is a good substrate prompted us to examine the possibility of other aromatic amino acids being transported by the LIV-I system. We found that the uptake of phenylalanine by the LIV-I system was inhibited in the presence of tyrosine with a Ki value of 120 µM (Table 2), suggesting the ability of the system to transport tyrosine. {Delta}tyrP YG228 was constructed (YG256), similarly transformed with pYG218 (livHMGF) and pYG237 (livJ), and then examined for transport. Low-level accumulation of labeled tyrosine was observed (Fig. 3A) but not in the strain carrying the empty vector. The Km value for tyrosine (230 µM) in the LIV-I system was comparable to the Ki value (200 µM) determined by its inhibition of leucine uptake. Although the accumulation of tyrosine was appreciable, the presence of the LIV-I/LS system alone could not support the growth of a tyrosine-auxotrophic strain ({Delta}aroP {Delta}brnQ {Delta}mtr {Delta}pheP {Delta}tna {Delta}tyrP {Delta}tyrA::kan+) in minimal medium even in the presence of 100 µM tyrosine, maybe due to the low affinity for tyrosine. Therefore, it seems likely that the LIV-I/LS system is not a physiologically important tyrosine transporter in E. coli. As for tryptophan, neither inhibition of leucine uptake nor accumulation in the cells by the LIV-I system was observed (10 to 300 µM) (data not shown). Alanine, serine, and threonine acted as inhibitors of phenylalanine transport by the LIV-I system (data not shown), as expected from the results obtained in binding studies with LIV-BP by Rahmanian et al. (38).

Similar experiments were performed with LS-expressing cells, and not only leucine but also isoleucine and valine were found to inhibit phenylalanine uptake. This was surprising, because it has been shown that purified LS-BP preferentially binds leucine (0.4 µM) but not isoleucine or valine (>1 mM each) (24). We carried out transport assays with labeled substrates (Fig. 3B) and found that the LS system was able to transport isoleucine and valine in addition to leucine. The DNA sequence of the livK gene on pYG239 was again analyzed, but no difference was found from the results reported by Blattner et al. (4). The Km values for isoleucine, leucine, and valine in the LS system were determined to be 5.0, 2.3, and 9.2 µM, respectively. There are apparent contradictions between the results of binding studies (14, 33, 40) and transport studies; is an auxiliary protein involved in the recognition of substrates by LS-BP or does the presence of membrane components LivHMGF alter the substrate specificity of LS-BP? In vitro uptake studies with the LS system reconstituted in liposomes are necessary to explain this discrepancy.

Of the aromatic amino acids tested (10 to 300 µM), only phenylalanine acted as a substrate for the LS system. Phenylalanine inhibited leucine uptake with a Ki value of 74 µM, which was comparable to the Km value of 30 µM. Likewise, the Ki values estimated for isoleucine (6.6 µM), leucine (2.1 µM), and valine (2.7 µM) in inhibition assays of phenylalanine uptake were in good accordance with the Km values obtained for them (5.0, 2.3, and 9.2 µM, respectively). The presence of alanine, serine, and threonine (each at 100 µM) did not affect phenylalanine uptake by the LS system at the saturating concentration.

Thus, consistent results were obtained in our transport studies, which revealed new aspects of the substrate specificity of the LIV-I and LS systems. The neutral amino acid ATP-binding cassette-type transport system (Nat) of Synechocystis sp. strain PCC 6803 has been identified by means of insertional mutagenesis, and it was shown that the strain inactivated for NatB, a periplasmic binding protein, leaked significant amounts of amino acids alanine, isoleucine, leucine, valine, and phenylalanine into the medium (27), indicating a role of the Nat system in the recapture of these amino acids. Although the LivJ (LIV-BP) and LivK (LS-BP) proteins of E. coli exhibit low levels (16%) of identity with NatB with respect to amino acid sequences, a similar substrate specificity was suggested, which may help us understand the mechanism underlying the substrate recognition by these proteins.

Functional distinction among the three phenylalanine uptake systems AroP, PheP, and LIV-I/LS. To obtain a better understanding of the LIV-I/LS system as the phenylalanine transporter, the physiological significance of the AroP, PheP, and LIV-I/LS systems was evaluated. A strain expressing one of the three transport systems was made Phe- (pheA18::Tn10) by P1 transduction and then streaked onto an M63-glucose minimal medium plate containing phenylalanine (MMF) and onto an MMF plate including isoleucine, tryptophan, or tyrosine (MMF+I, MMF+W, or MMF+Y) (Fig. 4A). In parallel, a Phe- strain possessing either all or none of the phenylalanine transporters was constructed and streaked onto similar plates. A phenylalanine transport-deficient Phe- strain was obtained by spreading the transductants [YG201 and P1(NK6024)] on LB plates containing 1 mM phenylalanine. Since phenylalanine accumulation could not be detected in the YG74 ({Delta}aroP {Delta}livHMGF {Delta}pheP) cells, even in the presence of 100 µM phenylalanine (data not shown), it seems likely that nonspecific diffusion of phenylalanine at the high concentration (1 mM) can support the growth of the strain.



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  		FIG. 4. Physiological role of each phenylalanine transport system in E. coli cells grown under various conditions. (A) AroP-, BrnQ-, LIV-I/LS-, and PheP-expressing Phe- strain YG208 (All), LIV-I/LS-expressing Phe- strain YG210 (LIV-I/LS), PheP-expressing Phe- strain YG211 (PheP), AroP-expressing Phe- strain YG212 (AroP), and Phe- strain YG213 not carrying any of these transport systems (None) were streaked on M63-glucose minimal medium plates containing 100 µM phenylalanine (MMF) and on MMF supplemented with 1 mM isoleucine (MMF+I), tryptophan (MMF+W), or tyrosine (MMF+Y). (B) Accumulation of phenylalanine in E. coli cells with various phenylalanine transport systems in the presence of 1 mM tyrosine. Strains MG1655 ({square}), YG106 (LIV-I/LS) ({circ}), YG108 (PheP) (•), and YG109 (AroP) ({blacksquare}) were grown in MMF+Y, and after the optical density at 600 nm had reached 0.5, the cells were harvested and suspended in MMF+Y containing 60 µg of chloramphenicol/ml. Cell suspensions were incubated in the presence of 100 µM labeled phenylalanine, and samples were withdrawn at the times indicated. The experiments were repeated three times with essentially the same results; the data for a representative experiment are shown.

 
As shown in Fig. 4A, growth of the Phe- strain in the presence of phenylalanine was dependent on the presence of any one of the AroP, PheP, or LIV-I/LS systems and no growth was observed for the Phe- strain lacking them. The same results were obtained regardless of the presence or absence of the BrnQ system (data not shown). The growth rates of the strains carrying the respective phenylalanine transporters did not differ significantly. These results confirm the physiological importance of these three transporters in phenylalanine accumulation.

The addition of isoleucine, which is a good substrate for both the LIV-I and LS systems, to the MMF medium severely inhibited the growth of LIV-I/LS-expressing Phe- strain YG210 ({Delta}aroP {Delta}brnQ {Delta}pheP pheA18::Tn10), whereas the growth of the AroP- and PheP-expressing Phe- strains was not affected. Likewise, the presence of tryptophan or tyrosine, either of which acts as a competitor for phenylalanine transport in the AroP system, caused significant retardation of the growth of AroP-expressing Phe- strain YG212 ({Delta}brnQ {Delta}livHMGF {Delta}pheP pheA18::Tn10) on the MMF+W or +Y medium. The inhibitory effect was greater for tryptophan than for tyrosine. The same results were obtained for five independently constructed strains. This was surprising, because the expression of the aroP gene is known to be strongly repressed by tyrosine but not by tryptophan (9, 22, 36, 46-48, 55) and the AroP system is known to exhibit almost equal affinity for the three aromatic amino acids (5, 36). At present, the reason for this phenomenon is unclear. PheP-expressing Phe- strain YG211 ({Delta}aroP {Delta}brnQ {Delta}livHMGF pheA18::Tn10) grew well under all conditions tested (PheP). Although the PheP and LIV-I systems are capable of transporting tyrosine, no inhibitory effect was observed in the presence of tyrosine (MMF+Y), reflecting the high Km values for tyrosine compared to those for phenylalanine in these systems.

Transport studies were carried out using these cells grown under the same conditions, and the results were consistent with the growth behavior shown in Fig. 4A. It is notable that when cells expressing the individual phenylalanine transport systems were grown in MMF+Y and then assayed for transport (Fig. 4B), the LIV-I/LS-expressing cells exhibited the highest phenylalanine uptake activity, which was almost the same as that of wild-type strain MG1655. Similar results were obtained when these cells were grown in MMF+W (data not shown). Thus, in the presence of tryptophan or tyrosine, the LIV-I/LS system plays a major role in phenylalanine accumulation in E. coli cells.

In conclusion, the LIV-I/LS system was identified as the third phenylalanine transporter in E. coli, which plays a significant role in the accumulation of phenylalanine in cells, especially when grown in the presence of tryptophan or tyrosine. The substrate specificities of the LIV-I and LS systems revealed by transport studies contradicted those found previously in binding studies; the reason for this contradiction remains to be elucidated.


   ACKNOWLEDGMENTS
 
This work was partly supported by a Grant-in-Aid for Scientific Research (B), no. 14360056, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, by a Grant-in-Aid for Fine Enzymatic Synthesis of Useful Compounds from Research for the Future (RFTF) of the Japan Society for the Promotion of Science, and by a Grant-in-Aid for Aromatic Amino Acid Metabolism in Bacteria from the Noda Institute for Scientific Research. T. Koyanagi is supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81-75-753-6276. Fax: 81-75-753-6275. E-mail: hidekuma{at}kais.kyoto-u.ac.jp. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Journal of Bacteriology, January 2004, p. 343-350, Vol. 186, No. 2
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.2.343-350.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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