Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli

Abstract

The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP-lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae.

Molecular analysis of the TyrR protein-mediated activation of mtr gene expression in Escherichia coli K-12

“…173,1991 on May 7, 2018 by guest http://jb.asm.org/ Downloaded from are low, the presence of phenylalanine in the medium results in enhanced expression of both mtr and tyrP, whereas tyrosine causes enhancement only in the case of mtr. By contrast the gene for the phenylalanine-specific transport system (pheP) does not appear to be subject to any specific regulation of activity (20), and the gene for the general aromatic amino acid transport system (aroP) is repressed by high intracellular pools of phenylalanine, tyrosine, and tryptophan (6,27 …”

Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli

“…1) (8,14,25). This raised the possibility that these residues may in some way contribute to a channel for the transport of phenylalanine or tyrosine (14).…”

“…A putative topological model involving 12 membrane spans has been proposed for PheP (14). In this model, one face of putative span III contained a contiguous array of aromatic amino acids.…”

“…The Mtr and TnaB proteins transport only tryptophan, PheP and TyrP selectively transport phenylalanine and tyrosine, respectively, and the AroP protein transports all three amino acids. Comparison of the coding sequences for each of these proteins puts them in two groups (14,19). PheP and AroP are highly homologous (60.4% identity) and are part of a group of prokaryotic amino acid transporters that also includes GabP, LysP, and a yet-unidentified protein, YifK (3,8,12,14,20).…”

“…Comparison of the coding sequences for each of these proteins puts them in two groups (14,19). PheP and AroP are highly homologous (60.4% identity) and are part of a group of prokaryotic amino acid transporters that also includes GabP, LysP, and a yet-unidentified protein, YifK (3,8,12,14,20). This group, together with a number of yeast membrane proteins also involved in the transport of amino acids (Canl, Gapl, Ycc5, Put4, and Hipl [6,7,9,21,22]), forms a cluster in a proposed family of transporters for amino acids, polyamines, and choline (16).…”