Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

Abstract

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 (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). 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 K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, 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.

FIG. 1.

Inhibition of phenylalanine uptake by branched-chain amino acids. (A) Growth inhibition of phenylalanine-auxotrophic (Phe⁻) E. coli strain TK1173 (ΔaroP Δmtr ΔpheP Δtna ΔtyrP 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 (ΔaroP Δmtr ΔpheP Δtna ΔtyrP). l-Phenylalanine was added to cell suspensions to a final concentration of 50 μM in either the absence (□) or presence of 5 μM glutamate (○), leucine (▪), 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.

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 (ΔaroP ΔlivHMGF ΔpheP) (BrnQ) (▴), YG106 (ΔaroP ΔbrnQ ΔpheP) (LIV-I/LS) (□), YG108 (ΔaroP ΔbrnQ ΔlivHMGF) (PheP) (•), YG109 (ΔbrnQ ΔlivHMGF ΔpheP) (AroP) (▵), and YG201 (ΔaroP ΔbrnQ ΔlivHMGF ΔpheP) (○) cells, and compared to that in wild-type strain MG1655 (▪). 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.

FIG. 3.

Uptake studies of various amino acids with E. coli cells expressing the LIV-I (A) and LS (B) systems. (A) Strain YG228 [ΔaroP ΔbrnQ Δ(livJ-yhhK-livKHMGF) ΔpheP] 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 [ΔaroP ΔbrnQ Δ(livJ-yhhK-livKHMGF) ΔpheP ΔtyrP] was used instead of YG228. Cell suspensions were incubated in the presence of 10 to 300 μM l-phenylalanine (□, ▪) and 25 to 300 μM l-tyrosine (▿, ▾). (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 (□, ▪), 0.4 to 70 μM l-leucine (⋄, ♦), l-isoleucine (○, •), or l-valine (▵, ▴). All experiments were repeated three times with essentially the same results; the data for a representative experiment are shown.

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 (□), YG106 (LIV-I/LS) (○), YG108 (PheP) (•), and YG109 (AroP) (▪) 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.