Introduction

Since its isolation, Corynebacterium glutamicum has been playing a central role in the developments of new knowledge and technology for various amino acid productions (Burkovski 2008; Eggeling and Bott 2005; Jetten et al. 1994; Kinoshita et al. 1957). Stimulated by the accessibility of the C. glutamicum genome (Ikeda and Nakagawa 2003; Kalinowski et al. 2003), this bacterium has also been used as a model for Gram-positive actinobacteria to understand microbial metabolism of aromatic compounds in our lab. A novel mycothiol-dependent gentisate catabolic pathway (Feng et al. 2006) and a link between aromatic degradation and gluconeogenesis for cell growth (Qi et al. 2007) were discovered. Regulations of aromatic metabolism in this strain were also investigated recently, and a novel atypical Lux family regulator was identified (Zhao et al. 2010). Those studies invoked the idea that aromatic compounds, such as derivatives of lignin, are potential substrates for production of amino acids. Recently, Lee et al. (2010) demonstrated that phenol was converted to glutamate and proline by C. glutamicum.

The robust ability of C. glutamicum to grow on a variety of aromatic compounds (Shen et al. 2004, 2005) relies on its multiple transporters for uptake of aromatic compounds. Genome data mining and experimental results confirmed that C. glutamicum had five transporters, i.e., the BenE/BenK, PcaK, VanK, and GenK, which were respectively responsible for the uptake of benzoate, protocatechuate, vaniliate, and gentisate (Chaudhry et al. 2007). A putative transporter (NCgl2953) located at downstream of resorcinol degradative genetic cluster (ncgl2950–ncgl2952) was proved to be a myo-inositol transporter (IolT2) (Krings et al. 2006), and it was not involved in resorcinol transport. Another putative transporter gene (ncgl1108) was located at the upstream of the regulator-encoding gene (ncgl1110) for resorcinol degradation (Huang et al. 2006). This invoked our interest to investigate the function of ncgl1108 in C. glutamicum. In this study, gene disruption/complementation and 14C-labeled aromatic amino acid uptake assays were carried out to identify the function of this putative transporter gene. It turned out that the gene ncgl1108 was involved in the uptake of l-Phe but not in resorcinol uptake or degradation.

Materials and methods

Bacterial strains, growth conditions, and plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. All Escherichia coli strains were grown in Luria–Bertani (LB) broth aerobically on a rotary shaker (200 rpm) at 37 °C or on LB plates with 1.2% (w/v) agar. C. glutamicum strains were routinely grown at 30 °C on a rotary shaker (200 rpm) in LB broth. To evaluate the growth of C. glutamicum strains on resorcinol and various aromatic amino acids, minimal medium (Konopka 1993) was supplemented with 2 mM resorcinol, l-Phe, l-Trp, or 1.5 mM L-Tyr as carbon or nitrogen source. Cell growth was monitored by measuring the turbidity at a wavelength of 600 nm (OD600). Antibiotics were used at the following concentrations: kanamycin, 50 μg ml−1 for E. coli and 25 μg ml−1 for C. glutamicum; ampicillin, 100 μg ml-1 for E. coli; chloramphenicol, 20 μg ml−1 for E. coli and 10 μg ml−1 for C. glutamicum.

Table 1 Bacterial strains, plasmids, and primers used in this study
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DNA extraction and manipulation

The total genomic DNA of C. glutamicum was isolated according to Tauch et al. (1995). DNA restriction enzyme digestion, plasmid isolation, and agarose gel electrophoresis were carried out as described previously (Sambrook et al. 1989). Plasmids were transformed into E. coli and C. glutamicum by electroporation (Tauch et al. 2002).

Amplification of DNA fragments with PCR and construction of plasmids

PCRs were performed by using Pfu DNA polymerase or Taq DNA polymerase (Takara, Japan). The PCR products were purified by using agarose gel DNA fragment recovery kit (Sangon, China). Cloning of PCR fragments was performed with the pMD19-T simple cloning vector system (Takara, Japan). Five plasmids for genetic disruption (pGXKZ4 and pGXKZ5), complementation (pGXKZ1), and gene expression (pGXKZ2 and pGXKZ3) in E. coli and C. glutamicum were constructed with pK18mobsacB or pXMJ19 (Table 1). The primers used for amplification of the intact or disrupted target gene fragments are listed in Table 1. For gene expression and genetic complementation, the pGXKZ1 was constructed by the insertion of the PCR-amplified intact gene, ncgl1108, into pXMJ19. The pGXKZ2 was constructed by insertion of the PCR-amplified gfp from pAcGFP into pXMJ19 and was used as a reference for cellular localization of NCgl1108. The pGXKZ3 was constructed by consecutively cloning of ncgl1108 (stop codon was deleted) and the SalI/EcoRI gfp fragment from pAcGFP into pXMJ19. In vitro disruption of ncgl1108 or aroP Cg was performed by removal of its partial region through restriction enzyme digestion. The full lengths of the intact ncgl1108 and aroP Cg were 1,407 and 1,392 bp, respectively. pGXKZ4 was constructed by cloning the disrupted ncgl1108 (the fragment from 255 to 971 bp was removed with StyI digestion) into pK18mobsacB. pGXKZ5 was constructed by cloning the disrupted aroP Cg (the fragment from 448 to 1057 bp was removed with ScaI digestion) into pK18mobsacB.

Genetic disruption and complementation in C. glutamicum

The pK18mobsacB derivatives were transformed into C. glutamicum RES167 by electroporation (Tauch et al. 2002). Screening for the first and second recombination events, as well as confirmation of the chromosomal deletion, was performed as described previously (Schafer et al. 1994). The resulting strains were designated C. glutamicum RES167∆ncgl1108, RES167∆aroP Cg, and RES167∆(ncgl1108-aroP Cg) (Table 1). The deletion of the target genes in pK18mobsacB derivatives and in C. glutamicum mutants was verified by PCR amplification and DNA sequencing. The gene expression in C. glutamicum was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to culture media.

Assays for aromatic amino acid transport

Uptake assays with unlabeled l-Phe, l-Tyr, and l-Trp. C. glutamicum RES167 cells were grown in LB medium. Cells at exponential phase were harvested and washed with ammonium-free minimal medium CGXII containing 0.1 M glucose and supplemented with 0.2 mg l−1 thiamine (Keilhauer et al. 1993). The harvested cells were resuspended (cell density OD600 of 8–10) in this CGXII medium containing 1 mM l-Phe, l-Tyr, or l-Trp and were incubated at 30 °C. At the indicated intervals, portions of the reaction mixture were withdrawn, filtered, and analyzed with HPLC (Yang et al. 2003). Uptake assay with 14C-labeled l-Phe, l-Tyr, or l-Trp. C. glutamicum RES167 and its mutants were cultivated and harvested according to the above described procedures. Cells were washed twice with 0.1 M Tris phosphate buffer (pH 6.8) and resuspended in the same buffer. The uptake of aromatic amino acids was measured by 14C-labeling liquid scintillation counting (Ikeda and Katsumata 1994) with the following modifications. The reaction mixture (1 ml) contained 100 μmol Tris phosphate (pH 6.8), 1 μmol MgSO4, 10 μmol glucose, 100 μg chloramphenicol, and 0.1 ml of the cell suspension (approximately 0.2 mg dry cells). The reaction was started by the addition of l-[14C(U)]-Phe, l-[side chain-3-14C]-Trp (PerkinElmer, Inc., USA), or l-[14C(U)]-Tyr (ARC, Inc., USA). At the indicated intervals, 50 μL of the reaction mixture was withdrawn, vacuum-filtered using nitrocellulose filters with a pore size of 0.22 μm, and immediately washed two times with 2 ml portions of cold 0.1 M LiCl. The filters containing cells were put into 2.0 ml centrifuge tubes filled with scintillation liquid. Radioactivity was determined by a PerkinElmer MicroBeta Liquid Scintillation counter. In order to obtain the uptake kinetics, 1–50 μM of 14C-l-Phe was applied, and the uptake rates for the first 60 s were determined. The uptake kinetics and activity was expressed as nanomoles of amino acid taken up per milligram of dry cell weight.

For export assay, the cells were cultivated and harvested as above described, but suspended (cell density OD600 of 2.0) in ammonium-free CGXII containing 1 mM tri-peptide (Phe–Phe–Phe, Tyr–Tyr–Tyr, or Trp–Trp–Trp; Sangon Biotech, Shanghai). This cell suspension was incubated at 30 °C for 2 h. Then, the cells were harvested and washed with ammonium-free CGXII solution. Cells were again suspended (cell density OD600 was 8–10) in ammonium-free CGXII containing 1 mM tri-peptide. The cells were incubated at 30 °C. At the indicated intervals, portions of the reaction mixture were withdrawn and filtered. The extracellular aromatic amino acid concentration was quantified by HPLC (Yang et al. 2003). The intracellular aromatic amino acid concentration was determined according to the procedures described by Simic et al. (2001).

Data analysis and statistics

The data obtained from uptake assays were analyzed with Microsoft Office Excel 2007. The differences of uptake (amounts) between wild type and mutants were expressed as averages of all determinations at the time period specified in this study.

Cellular localization of NCgl1108-GFP fusion proteins with confocal microscopy

The localization of NCgl1108-GFP was conducted according to Xu et al. (2006). Specifically, plasmid pGXKZ2 and pGXKZ3 were transformed into competent E. coli DH5α and C. glutamicum RES167 by electroporation. The recombination strains were incubated overnight in LB broth. When the culture OD600 reached approximately 0.5, IPTG was added to a final concentration of 0.1 mM. Cells were harvested and washed twice and suspended in 0.9% sodium chloride. This cell suspension was mixed with agarose (final concentration of 0.24%). Samples of the cell–agarose mixture were imaged under confocal microscope with excitation filter 475 nm and emission filter 505 nm. The imaging experiments were performed using a Leica TCS SP2 laser scanning spectral confocal microscope equipped with a cooled CCD camera.

Results

Bioinformatic analyses of ncgl1108 and its translational product (NCgl1108)

The gene ncgl1108 was located at upstream of the previously characterized resorcinol gene cluster (ncgl1110–ncgl1113) (Huang et al. 2006). It encodes a hypothetical protein of 468 amino acid residues with a calculated molecular mass of 50.2 kDa. BLAST-P searches showed that the NCgl1108 had 51% sequence identity to the ProYSt (l-Pro-specific permease) of Salmonella typhimurium (Liao et al. 1997). In addition, NCgl1108 showed 36% identity to the AroPCg (general aromatic amino acid transporter) of C. glutamicum (Wehrmann et al. 1995). Other proteins that showed significant identities to NCgl1108 were AroPEc (Chye et al. 1986), and PhePEc (l-Phe-specific transporter, 41%; Pi et al. 1991). NCgl1108 was predicted to be a membrane protein with 12 transmembrane helices, and alignment of NCgl1108 to its analogous transporters revealed that it possessed the signature sequences of the AAT family of APC superfamily (Jack et al. 2000). Previously, Marin and Krämer (2007) predicted that NCgl1108 (Cgl1155 or Cg1305) coded for an APC-type carrier of unknown substrates, and this ncgl1108 was annotated later as a putative proline permease (http://www.membranetransport.org). Our analyses suggested that ncgl1108 was possibly involved in resorcinol, l-Pro, l-Tyr, l-Phe, and/or l-Trp transport.

Genetic disruption of ncgl1108 affected the growth of C. glutamicum on l-Phe, but not on resorcinol, l-Pro, l-Tyr, and l-Trp

In order to investigate its function, ncgl1108 was disrupted in C. glutamicum RES167, resulting in the mutant RES167∆ncgl1108 (Table 1). The RES167∆ncgl1108 and RES167 were cultivated in LB and minimal media with resorcinol as carbon source, and no phenotypic differences were observed. This result ruled out our hypothesis that ncgl1108 was involved in resorcinol metabolism although it neighbored the resorcinol gene cluster.

Genome data mining with KEGG pathway tool showed that C. glutamicum had incomplete metabolic pathways for l-Pro, l-Phe, l-Tyr, or l-Trp, indicating that C. glutamicum RES167 was not able to grow on them as carbon source. Our experiments confirmed this genome-mining result: C. glutamicum did not grow on l-Pro, l-Phe, l-Tyr, or l-Trp as carbon source. However, we found that those amino acids could support the growth of RES167 when they were served as sole nitrogen sources, although the biomass accumulation was not high (Fig. 1a–d). Genetic disruption of ncgl1108 did not affect the growth of RES167∆ncgl1108 on l-Tyr (Fig. 1a), l-Pro (Fig. 1b), or l-Trp (Fig. 1c), but impaired its growth on l-Phe (Fig. 1d). Genetic complementation of ncgl1108 in RES167∆ncgl1108/pGXKZ1 restored its growth on l-Phe (Fig. 1d).

Fig. 1
figure 1

Growth of C. glutamicum RES167 (filled square), RES167Δncgl1108 (empty triangle), and RES167Δncgl1108/pGXKZ1 (filled triangle) on minimal medium supplemented with l-Tyr (a), l-Pro (b), l-Trp (c), or l-Phe (d) as sole nitrogen source

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Disruption and hyperexpression of ncgl1108 significantly affected the uptake of l-Phe by C. glutamicum cells

Combining the bioinformatic analyses and the above experimental results, it was deduced that the gene ncgl1108 encoded a putative l-Phe transporter. Uptake assays for l-Phe, l-Tyr, l-Pro, or l-Trp by wild RES167 and mutant RES167∆ncgl1108 were conducted. The results showed that the disruption of pheP Cg resulted in differences for l-Tyr, l-Pro, or l-Trp uptake between RES167 and RES167∆ncgl1108 (Fig. 2; white and gray columns). Statistical analysis showed that uptakes of l-Phe, l-Tyr, l-Pro, and l-Trp by RES167∆ncgl1108 decreased by 18.2 ± 4.9%, 0.6 ± 2.7%, 6.8 ± 10.4%, 6.0 ± 6.3%, respectively, when compared to RES167. In order to characterize the effect of NCgl1108 on l-Phe uptake further, the ncgl1108 was hyperexpressed with multicopy pGXZ1 in RES167 cells. Compared to RES167 and mutant RES167∆ncgl1108, this hyperexpression of ncgl1108 in RES167/pGXKZ1 resulted in significant increase (104.0 ± 29% in average) of l-Phe uptake (Fig. 2d; black columns). The effects of hyperexpression of ncgl1108 in RES167/pGXKZ1 on l-Tyr, l-Trp, or l-Pro uptake were also observed, but not so significant. Based on these results, it is concluded that ncgl1198 encodes an l-Phe transporter and is named as pheP Cg.

Fig. 2
figure 2

Uptake of l-Tyr (a), l-Trp (b), l-Pro (c), or l-Phe (d) by C. glutamicum RES167 (gray column), RES167Δncgl1108 (white column), and RES167/pGXKZ1 (black column)

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To determine if PhePCg functioned as an exporter for l-Phe, l-Tyr, or l-Trp, export experiments with tri-peptides (Phe–Phe–Phe, Tyr–Tyr–Tyr, or Trp–Trp–Trp) were carried out. The results revealed that the bulk concentrations of l-Phe, l-Tyr, and l-Trp in experiments with RES167/pGXKZ1 were not higher than that with RES167, indicating that the PhePCg did not have export function (Fig. 3a–c). It is noteworthy that the bulk concentrations of l-Phe in experiment with RES167/pGXKZ1 were even lower than that with RES167 (Fig. 3a). Further studies showed that the intracellular l-Phe levels in RES167/pGXKZ1 and in RES167 were higher than that in RES167∆ncgl1108 (Fig. 3d), indicating the accumulation of l-Phe caused by the occurrence of PhePCg in RES167/pGXKZ1 and RES167 cells. These results supported that PhePCg functioned as an importer for l-Phe.

Fig. 3
figure 3

Export assays for l-Phe (a), l-Tyr (b), or l-Trp (c), and intracellular concentration for LPhe (d) by C. glutamicum RES167 (filled square), RES167Δncgl1108 (empty triangle) and RES167Δncgl1108/pGXKZ1 (filled triangle) in minimal medium containing 1 mM of Phe–Phe–Phe (a, d), Tyr–Tyr–Tyr (b) or Trp-Trp-Trp (c)

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Construction of the double mutant RES167∆(ncgl1108-aroP Cg) and determination of uptake kinetics for aromatic amino acids

The previously identified AroPCg uptakes all three aromatic amino acids in C. glutamicum (Wehrmann et al. 1995). In order to eliminate the effect of AroPCg on aromatic amino acid uptake assay and to estimate the uptake kinetics of PhePCg, we constructed a double mutant, RES167∆(ncgl1108-aroP Cg), by further disruption of aroP Cg in RES167∆ncgl1108 in this study (Table 1). Difference in growth in LB medium among RES167∆(ncgl1108-aroP Cg), RES167∆ncgl1108, and RES167 was not observed.

The uptake of 14C-labeled l-Phe or l-Tyr was determined with wild recombinant strains and mutants. The uptake of 14C-labeled l-Phe by mutants RES167∆ncgl1108 and RES167∆(ncgl1108-aroP Cg) was significantly lower compared to the RES167 (Fig. 4a). Statistical analysis of these data revealed that the uptakes of l-Phe by mutants RES167∆ncgl1108 and RES167∆(ncgl1108-aroP Cg) decreased by 13.2 ± 1.9% and 39.8 ± 1.8%, respectively. Hyperexpression of PhePCg in RES167∆(ncgl1108-aroP Cg)/pGXKZ1 resulted in 47.6 ± 6.8% increase of l-Phe uptake. The effect of PhePCg disruption on 14C-labeled l-Tyr uptake was much less significant (Fig. 4b). Compared to RES167, the uptake for l-Tyr by mutant RES167∆ncgl1108 decreased by 7.8 ± 3.1%. Determination of l-Phe uptake kinetics of RES167∆(ncgl1108-aroP Cg)/pGXKZ1 (Fig. 4c) showed that its K m and V max values were 10.4 ± 1.5 μM and 1.2 ± 0.1 nmol min−1 (mg DW)−1, respectively. The K m and V max values of RES167∆(ncgl1108-aroP Cg) for l-Phe were determined to be 4.0 ± 0.4 μM and 0.6 ± 0.1 nmol min−1 (mg DW)−1, respectively. The higher V max value of RES167∆(ncgl1108-aroP Cg)/pGXKZ1 than that of RES167∆(ncgl1108-aroP Cg) clearly indicated that PhePCg was active and functional in RES167∆(ncgl1108-aroP Cg)/pGXKZ1 at the conditions tested in this study. The kinetic analysis also revealed that additional l-Phe transporter(s) besides aroP Cg and PheP Cg still occurs in the double mutant RES167∆(ncgl1108-aroP Cg).

Fig. 4
figure 4

Uptake of 14C-labeled l-Phe (a) and l-Tyr (b) by C. glutamicum RES167 (filled square), RES167Δncgl1108 (empty triangle), RES167Δ(ncgl1108-aroPCg) (empty diamond), and RES167Δ(ncgl1108-aroP)/pGXKZ1 (filled diamond), and determination of K m and V max for l-Phe (c) by C. glutamicum RES167Δ(ncgl1108-aroPCg)/pGXKZ1. The initial concentrations of 14C-labeled l-Phe was 50 μM (a, b) and 1–50 μM (c). The K m and V max values of C. glutamicum RES167Δ(ncgl1108-aroPCg)/pGXKZ1 and C. glutamicum RES167Δ(ncgl1108-aroPCg) for l-Phe were obtained by use of the experimental data shown in c and by conversion of those data into Lineweaver–Burk plots

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The substrate specificity of PhePCg in C. glutamicum RES167∆(ncgl1108-aroP)/pGXKZ1 was examined with 14C-labeled l-Phe in the presences of 20-fold unlabeled various amino acids (Table 2). Results indicated that PhePCg was specific to l-Phe, and its transport activity for l-Phe was not affected by all tested amino acids, except for l-Tyr (Table 2). The uptake of 14C-labeled l-Phe was strongly inhibited by l-Tyr was surprising. We deduced that the inhibition by l-Tyr was due to the structural similarity between l-Phe and l-Tyr.

Table 2 Effects of various amino acids on l-Phe uptake in C. glutamicum RES167∆(ncgl1108-aroP)/pGXKZ1
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PhePCg was localized at cellular membrane

In order to identify the cellular localization of PhePCg, a fusion protein was engineered from the PhePCg and GFP. For the purpose to ensure correct folding of peptides, a 16-amino acid-long linker was installed between the PhePCg and GFP peptides, so that each of them was still folded correctly and functioned individually. The fusion protein PhePCg-GFP was synthesized under induction with IPTG in cells of C. glutamicum RES167/pGXKZ3. Confocal microscopy clearly showed that the fusion protein PhePCg-GFP was located at the cellular periphery membrane part of C. glutamicum RES167/pGXKZ3 (Fig. 5).

Fig. 5
figure 5

Confocal microscopy of C. glutamicum RES167/pGXKZ2 (control) and C. glutamicum RES167/pGXKZ3. Cells were cultivated as described in “Materials and methods”, and were induced with IPTG. Fusion protein of PhePCg-GFP in C. glutamicum was visualized by its fluorescence. C. glutamicum RES167/pGXKZ2 under fluorescence (a) and visible light (b) and C. glutamicum RES167/pGXKZ3 under fluorescence (c) and visible light (d)

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Discussion

The transport of aromatic acids into cells is the first step for bacterial metabolism of these compounds. In C. glutamicum, the transporter genes involving in aromatic compound metabolism such as genK and benK/benE often associate with the degradative gene clusters (Chaudhry et al. 2007). Although the gene pheP Cg (ncgl1108) is located immediately upstream of the resorcinol degradative gene cluster, our results demonstrated that this gene was not involved in resorcinol degradation. Instead, pheP Cg encodes an l-Phe specific transporter in C. glutamicum.

So far, as we know, PhePCg is the first l-Phe specific transporter identified from C. glutamicum, and it represents the first functionally identified l-Phe specific transporter from Gram-positive bacteria. Early studies suggested that l-Phe and l-Tyr were transported in Bacillus subtilis by a common system (D’Ambrosio et al. 1973); however, any l-Phe specific transporter has not been identified. In E. coli, three l-Phe transport systems were identified, i.e., the general aromatic amino acid transporter AroPEc that transports all three aromatic amino acids (Chye et al. 1986; Honore and Cole 1990), the l-Phe specific transporter PhePEc that is similar to PhePCg and they share 36% identity of amino acid sequence (Pi et al. 1991), and the branched-chain amino acid transport system LIV-I/LS system that functions as l-Phe transport (Koyanagi et al. 2004). Compared to other l-Phe transport systems, the RES167/PhePCg cells have moderate affinity to l-Phe: It has higher affinity than that of the E. coli/LIV-IEc (K m = 19 μM; Koyanagi et al. 2004) and Neurospora crassa/PhePNc (K m = 100 μM; DeBusk and DeBusk 1965), but much lower affinity when compared to that of the E. coli/AroPEc (K m = 0.47 μM; Brown 1970) and E. coli/PhePEc (K m = 2 μM; Brown 1970; Cosgriff et al. 2000). We observed that the occurrence of unlabeled l-Tyr significantly decreased the uptake of 14C-labeled l-Phe by PhePCg. As determined in this study, uptake of l-Tyr by PhePCg was not obvious in this study. This substrate spectrum of PhePCg is clearly different from the previously identified AroPCg from C. glutamicum (Wehrmann et al. 1995).

Based on our observation, the PhePCg is physiologically active and plays a role in uptake of l-Phe by C. glutamicum under the conditions examined in this study. Disruption of pheP Cg resulted in significant decreases of l-Phe uptake. Phenotypically, this disruption of pheP Cg reduced the growth of C. glutamicum when l-Phe served as sole nitrogen source. Exploitation of the C. glutamicum genome with KEGG pathway tools revealed that this bacterium is possibly able to assimilate l-Phe as nitrogen source and supports our observation that C. glutamicum grew on l-Phe as nitrogen source. Two candidate genes, ncgl0215 and ncgl2020, which encode putative l-Phe /l-Tyr aminotransferases and are possibly involved in deamination of l-Phe, were identified. It is proposed that the uptake of l-Phe by PhePCg increased the intracellular l-Phe concentration and subsequently invoked the activation of the putative aminotransferases in C. glutamicum. This physiological adaptation enables C. glutamicum growing on the l-Phe as sole nitrogen source. However, the growth was very limited when l-Phe served as sole nitrogen sources. Accumulation of phenylpyruvate, a deduced metabolite from l-Phe deamnination, was observed (data not shown).

It is deduced that other transport system(s), besides PhePCg and AroPCg, for l-Phe or other aromatic amino acids occur in C. glutamicum. This hypothesis is based on the observation that disruption of both PhePCg and AroPCg did not result in complete loss of l-Phe and other aromatic amino acid uptake. Genome-wide searches according to gene/amino acid sequence similarity revealed other putative aromatic amino acid transport genes, including ncgl0453 and ncgl0464. Functions of those putative transporter genes are currently under investigation.