Cloning, nucleotide sequence, and characterization of mtr, the structural gene for a tryptophan-specific permease of Escherichia coli K-12

Abstract

The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease. This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG.Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance. The mtr gene was cloned by using the mini-Mu system. The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP. The hydropathy plots of the two permeases were similar. Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter.In Escherichia coli, the aromatic amino acids are concentrated in the cytoplasm mainly via four operationally distinguishable transport systems. These systems are a general aromatic amino acid permease, encoded by aroP, and three other transport systems specific for phenylalanine, tyrosine, and tryptophan (6). The general aromatic amino acid permease has strong affinity for the aromatic amino acids (Km for all three amino acids, 10' M), whereas the individual transport systems have a lower affinity but a higher specificity for their respective aromatic amino acids (Km for each, ca. 10-6 M). Mutations in the structural genes for each specific permease have been found (6,19,39). The genes for the general aromatic amino acid permease, aroP, and the tyrosine-specific permease, tyrP, have been cloned (10,14) and sequenced (20,43). The regulation of transcription from the tyrP and aroP promoters has been thoroughly investigated (11,23,24). AroP and TyrP are hydrophobic proteins that are associated with the cytoplasmic membrane (10, 44).The tryptophan-specific transport system is encoded by a single gene designated mtr. This gene is situated near the 69-min region of the bacterial chromosome (19), and the locus was first identified through the isolation of mutations that impart resistance to the antimetabolite 5-methyltryptophan. By P1 transduction, mtr is 60 to 80% linked to argG (19,25). Mutations in mtr result in a loss of tryptophanspecific uptake (C. Yanofsky, cited by Oxender [34]). In addition to the aroP and mtr systems for L-tryptophan uptake, a gene for a low-affinity tryptophan permease (K, 10-5 M) is situated within the tna operon (14).This report describes the cloning and structural characterization of the mtr+ gene. The cloned DNA has structural and genetic properties predicted for a segment from the 69-min region of the chromosome. Several lines of evidence support the notion that the region cloned and sequenced in this study encodes the tryptophan-specific transport protein Mtr. Structural comparisons of Mtr with the two other wellcharacterized aromatic amino acid permeases of E. coli are also presented. Preparation of mini-Mu lysates. Mini-Mu lysates were prepared as described previously (18). MC1040-2 carrying plasmid pEG5005 was used to prepare a mini-Mu lysate. Lys...

Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli

“…In the studies described in this report we evaluated the contributions of the three permeases to tryptophan uptake by measuring tryptophan induction of the tna operon of E. coli. Considerable information is available on the regulation of the operons encoding the three transport proteins (5,9,10,11,22,25,28). Expression of the mtr operon is repressed by the trp repressor in excess tryptophan (10,11,22) and activated by phenylalanine and the tyrR product (22).…”

“…Considerable information is available on the regulation of the operons encoding the three transport proteins (5,9,10,11,22,25,28). Expression of the mtr operon is repressed by the trp repressor in excess tryptophan (10,11,22) and activated by phenylalanine and the tyrR product (22). tnaB, as the second gene in the tna operon, is subject to catabolite repression, and its expression is induced appreciably by tryptophan (7,9,25).…”

“…Tryptophan, in addition to its structural and functional role in proteins, can be degraded to indole, pyruvate, and ammonia, and the pyruvate and ammonia can be used as carbon and nitrogen sources (24). Three permeases mediate tryptophan uptake; they are designated Mtr, TnaB, and AroP (6,10,12,20,28). AroP also transports phenylalanine and tyrosine.…”

“…Mtr and TnaB are tryptophan specific, whereas AroP also transports phenylalanine and tyrosine. The genes for these permeases have been cloned and sequenced (4,6,10,13,21,22), and the sequences of their predicted polypeptide products suggest that each functions as a transmembrane protein (21 revealed that they are members of the same protein family (21); this family also includes the tyrosine-specific permease TyrP (10,21).…”

“…Tryptophanase can also catalyze the synthesis of tryptophan from indole and serine or cysteine (24). To achieve its tryptophan uptake objectives, E. coli synthesizes three tryptophan permeases, designated Mtr, TnaB, and AroP (2,3,7,10,12,17,20,22,28). Mtr and TnaB are tryptophan specific, whereas AroP also transports phenylalanine and tyrosine.…”