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 (⌬aroP ⌬pheP ⌬mtr ⌬tna ⌬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 K m values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 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.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)(35)(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 (⌬aroP ⌬pheP mtr24 ⌬tna ⌬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 accumu...

Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids

“…2B) when expressed in RU1357. The narrow specificity of the E. coli LivKHMGF system is consistent with a recent reinvestigation (9).…”

Selective Utilization of Exogenous Amino Acids by Dehalococcoides ethenogenes Strain 195 and Its Effects on Growth and Dechlorination Activity

“…Although data from the isotopomer analysis confirmed 195's ability to import amino acids with different chemical characteristics (see Table S3 in the supplemental material) (e.g., hydrophilic Thr, Lys, and His, together with hydrophobic Pro, Ala, Tyr, Val, Met, Leu, Ile, and Phe), the actual substrate(s) for the transporters have not yet been positively identified. For example, the predicted branched-chain transporter (DET0938, DET0941 to DET0944) was annotated to have a broad substrate specificity, and bioinformatics findings suggest that this transporter is similar to the LIV-I/LS system in E. coli (33), which catalyzes the translocation of not only Leu, Ile, and Val but also Phe and Tyr from the periplasm to the cytoplasm (18). An analogous broad specificity was observed in E. coli strain K-12 MG1655 (Fig.…”

Homocysteine Toxicity inEscherichia coliIs Caused by a Perturbation of Branched-Chain Amino Acid Biosynthesis

“…3) suggests that Hcy is not transported via either of these systems. To further test this conclusion, we compared the Hcy sensitivity of a wild-type E. coli strain (MG1655) to two isogenic mutants that were deficient for either of the two isoleucine uptake systems (⌬livH-MGF and ⌬brnQ [15]). All three strains were grown in MM with a range of Hcy concentrations (0 to 10 mM), and their growth rates were measured.…”