The tpl promoter of Citrobacter freundii is activated by the TyrR protein

Abstract

The ability of microorganisms to degrade L-tyrosine to phenol, pyruvate, and ammonia is catalyzed by the inducible enzyme L-tyrosine phenol lyase (EC 4.1.99.2). To investigate possible mechanisms for how the synthesis of this enzyme is regulated, a variety of biochemical and genetic procedures was used to analyze transcription from the tpl promoter of Citrobacter freundii ATCC 29063 (C. braakii). By computer analysis of the region upstream of the tpl structural gene, two segments of DNA bearing strong homology to the known operator targets of the TyrR protein of Escherichia coli were detected. A DNA fragment of 509 bp carrying these operator targets plus the presumptive tpl promoter was synthesized by PCR and used to construct a single-copy tpl-lacZ reporter system. The formation of ␤-galactosidase in strains carrying this reporter system, which was measured in E. coli strains of various genotypes, was strongly dependent on the presence of a functional TyrR protein. In strains bearing deletions of the tyrR gene, the formation of ␤-galactosidase was reduced by a factor of 10. Several mutationally altered forms of TyrR were deficient in their abilities to activate the tpl promoter. The pattern of loss of activation function was exactly parallel to the effects of the same tyrR mutations on the mtr promoter, which is known to be activated by the TyrR protein. When cells carrying the tpl-lacZ reporter system were grown on glycerol, the levels of ␤-galactosidase were 10-to 20-fold higher than those observed in glucose-grown cells. The effect was the same whether or not TyrR-mediated stimulation of the tpl promoter was in effect. By deleting the cya gene, it was shown that the glycerol effect was attributable to stimulation of the tpl promoter by the cyclic AMP (cAMP)-cAMP reporter protein system. A presumptive binding site for this transcription factor was detected just upstream of the ؊35 recognition hexamer of the tpl promoter. The transcriptional start point of the tpl promoter was determined by chemical procedures. The precise locations of the TyrR binding sites, which were established by DNase I footprinting, agreed with the computer-predicted positions of these regulatory sites. The two TyrR operators, which were centered at coordinates ؊272.5 and ؊158.5 with respect to the transcriptional start point, were independently disabled by site-directed mutagenesis. When the upstream operator was altered, activation was completely abolished. When the downstream operator was altered, there was a fourfold reduction in reporter enzyme levels. The tpl system presents a number of intriguing features not previously encountered in TyrR-activated promoters. First among these is the question of how the TyrR protein, bound to widely separated operators, activates the tpl promoter which is also widely separated from the operators.

Functional Analysis of the Erwinia herbicola tutB Gene and Its Product

“…Under tyrosine-induced conditions, the ␤-galactosidase activity of the cells carrying tutB was 1.3-fold as high as that of the cells not carrying tutB (4,400 against 3,300). The mechanism underlying TyrR-mediated tyrosine induction of tpl has been well established (14,28). These results indicated that introduction of the TutB system actually increased the intracellular level of tyrosine.…”

“…In a preliminary experiment, we observed that the introduction of pACYC177 carrying the tutB gene into an E. coli strain with the ⌽(tplЈ-Јlac) gene (14) enhanced the tyrosine-induction ratio of tpl by twofold, but neither the phenylalanine-nor the tryptophan-mediated induction ratio was elevated. Since the expression of tpl is positively regulated by the TyrR protein (22) and its coeffectors (three aromatic amino acids), even though the magnitude of activation is quite different among the three ligands (13,28), the above observation implied that the tutB gene might encode a tyrosine transporter. In order to characterize the tutB gene product, an E. coli strain deficient in the uptake of all aromatic amino acids (5) was constructed (MG1655 ⌬aroP mtr24 ⌬pheP ⌬tna ⌬tyrP) and used for transport assays.…”

Cloning and Random Mutagenesis of theErwinia herbicola tyrRGene for High-Level Expression of Tyrosine Phenol-Lyase

“…The regulatory mechanism underlying expression of tpl was investigated by means of the lac reporter system, and it was demonstrated that, at least in E. coli, both the TyrR protein and cyclic AMP receptor protein (CRP) participate in it (23,47). The TyrR protein plays a major role in the regulation of genes that are essential for the biosynthesis, transport, and degradation of aromatic amino acids (1,5,8,23,34,42,47).…”

Altered Oligomerization Properties of N316 Mutants ofEscherichia coliTyrR

“…Whereas hexamer formation of TyrR causes repression of aroF and tyrP, the tpl promoter is activated by ligand-mediated hexamerization of TyrR bound to the three distant boxes (Fig. 1A) (2,8,20). Activation of tpl also requires the binding of the aromatic amino acid to the N-terminal ATP-independent site.…”