Functional Analysis of the Erwinia herbicola tutB Gene and Its Product

Abstract

The tutB gene, which lies just downstream of tpl, has been cloned from Erwinia herbicola, and its product was analyzed. Despite its high sequence similarity to tryptophan transporters, TutB was found to be a tyrosinespecific transporter. Tryptophan acted as a competitive inhibitor of tyrosine transport. Unlike the tryptophanase operon, the tpl and tutB genes do not constitute an operon.Tyrosine phenol-lyase (Tpl) is a tyrosine-inducible enzyme distributed in some enteric bacteria (8). Tpl catalyzes the ␣,␤-elimination of L-tyrosine to produce pyruvate, ammonia, and phenol (16), thereby allowing bacteria to utilize L-tyrosine as carbon and nitrogen sources. One of the most notable features of Tpl is its ability to synthesize 3,4-dihydroxyphenylalanine (L-DOPA) from pyruvate, ammonia, and catechol through the reversal of ␣,␤-elimination (15). L-DOPA is used in the treatment of Parkinson's disease, which afflicts 1 out of every 1,700 individuals.During the course of study of tpl, an open reading frame, designated as tutB, was found just downstream of the tpl gene (9). It was predicted to encode a protein of 416 amino acids with many hydrophobic regions. Since the genetic organization of the tpl and tutB loci (Fig. 1A) is quite similar to that of the tryptophanase operon (tna consisting of tnaA [tryptophan indole-lyase] and tnaB [tryptophan-specific permease]) of Escherichia coli (7), and also since the physiological role of Tpl as to tyrosine is considered to be equivalent to that of TnaA as to tryptophan, the tutB gene was assumed to constitute an operon with tpl and to encode a tyrosine-specific transporter.In this study, we cloned the tutB gene from Erwinia herbicola and determined the properties of its product by using Escherichia coli cells. In addition, to elucidate the role of the TplTutB system in the tyrosine degradation pathway, lac fusions were constructed and their expression was monitored. The bacterial strains used in this study were derivatives of E. coli K-12 or E. herbicola AJ2985. The strains and plasmids are listed in Table 1 with their characteristics. Standard recombinant DNA procedures were used (23), and the results of in vitro manipulations such as PCR and mutagenesis were confirmed by DNA sequencing (24).Cloning and sequencing of tutB of E.herbicola. An open reading frame situated 155 nucleotides downstream of the coding region of tpl has already been found and designated tutB although its function has not been elucidated. A plasmid, pEBT, obtained in our previous study (31) contains the tpl gene and a C-terminally truncated tutB gene. The intact tutB gene was cloned from an E. herbicola genomic library constructed as described previously (13), and the resulting plasmid was named pTK928. The 0.6-kb MfeI-HpaI fragment excised from pTK928 and the 3.3-kb SmaI-MfeI fragment cut off from pEBT were annealed at their MfeI-protruding ends, and the resulting 3.9-kb fragment containing the entire tpl and tutB genes in their native configurations was cloned into the SmaI site of pUC19 to generate pTK1...

Identification of the LIV-I/LS System as the Third Phenylalanine Transporter in Escherichia coli K-12

“…The strains and plasmids are listed in Table 1 with their characteristics. The aroP, pheP, tna, and tyrP genes were disrupted as described previously (23), and the mtr gene was disrupted, using mtr-1 and mtr-2 (Table 1) as primers, by the method described by Datsenko and Wanner (12). Disruption of the brnQ gene was carried out as follows.…”

“…Transport assays. Transport assays were performed as described previously (23,51), with slight modifications as follows. Cells grown in minimal medium were harvested at mid-exponential phase and then washed twice with M63-glucose containing 60 g of chloramphenicol/ml to stop protein synthesis.…”

“…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.…”

“…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.…”

Altered Oligomerization Properties of N316 Mutants ofEscherichia coliTyrR

“…The aroF and tyrP genes of E. coli encode tyrosine-repressible 3-deoxyarabinoheptulosonate 7-phosphate synthase and tyrosinespecific permease, respectively (17)(18)(19), and the tpl gene of E. herbicola encodes tyrosine phenol lyase (6)(7)(8). In the cases of aroF and tyrP a strong box(es) is located upstream of, and a weak box overlaps with, the Ϫ35 promoter (Fig.…”