Synergism between the Trp repressor and Tyr repressor in repression of the aroL promoter of Escherichia coli K-12

Tryptophan is an essential amino acid that is required for normal development in Chlamydia species, and tryptophan metabolism has been implicated in chlamydial persistence and tissue tropism. The ability to synthesize tryptophan is not universal among the Chlamydiaceae, but species that have a predicted tryptophan biosynthetic pathway also encode an ortholog of TrpR, a regulator of tryptophan metabolism in many gram-negative bacteria. We show that in Chlamydia trachomatis serovar D, TrpR regulates its own gene and trpB and trpA, the genes for the two subunits of tryptophan synthase. These three genes form an operon that is transcribed by the major form of chlamydial RNA polymerase. TrpR acts as a tryptophan-dependent aporepressor that binds specifically to operator sequences upstream of the trpRBA operon. We also found that TrpR repressed in vitro transcription of trpRBA in a promoter-specific manner, and the level of repression was dependent upon the concentrations of TrpR and tryptophan. Our findings provide a mechanism for chlamydiae to sense changes in tryptophan levels and to respond by modulating expression of the tryptophan biosynthesis genes, and we present a unified model that shows how C. trachomatis can combine transcriptional repression and attenuation to regulate intrachlamydial tryptophan levels. In the face of host defense mechanisms that limit tryptophan availability from the infected cell, the ability to maintain homeostatic control of intrachlamydial tryptophan levels is likely to play an important role in chlamydial pathogenesis.

Section: Discussionmentioning

confidence: 99%

Molecular Mechanism of Tryptophan-Dependent Transcriptional Regulation inChlamydia trachomatis

Akers¹,

Tan

2006

“…The DNA sequences adjacent to the known tpl genes (3,15,22,25,39) were scrutinized for the presence of target sites for the TyrR protein. A consensus table based on authentic TyrR boxes was used as the reference system, as previously described (21). Several potential operator targets were detected in the region upstream of the tpl gene of C. freundii, the sequence of which was determined by Antson et al (3).…”

Section: Resultsmentioning

confidence: 99%

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

Smith

Somerville

1997

Self Cite

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.

“…The aroL-encoded enzyme has a K m of about 200 M for shikimate and appears to play a major role in the biosynthesis of the aromatic amino acids. Expression of the aroL gene is controlled by both the TyrR and TrpR proteins and the amino acids tyrosine and tryptophan as cofactors (3,5,6,8). The roles of aroK and shikimate kinase I are less clear.…”

mentioning

confidence: 99%

A reassessment of the relationship between aroK- and aroL-encoded shikimate kinase enzymes of Escherichia coli

Whipp

Pittard

1995

In the course of sequencing the aroK gene, a number of errors were found in the published sequence. The corrected sequence alters the length of the aroK coding region such that the AroK and AroL proteins are now of comparable length and the homology between them extends the entire length of the two enzymes.Escherichia coli synthesizes two shikimate kinase enzymes whose structures are encoded by the genes aroL for shikimate kinase II (3, 5) and aroK for shikimate kinase I (11). The aroL-encoded enzyme has a K m of about 200 M for shikimate and appears to play a major role in the biosynthesis of the aromatic amino acids. Expression of the aroL gene is controlled by both the TyrR and TrpR proteins and the amino acids tyrosine and tryptophan as cofactors (3,5,6,8). The roles of aroK and shikimate kinase I are less clear. Expression of the gene appears to be constitutive, and the enzyme has a much lower affinity for shikimate, with a reported K m of greater than 20 mM (4). The aroK gene was identified on the basis of the amino acid sequence homology between its putative product and the aroL-encoded protein. Its identity was confirmed by the construction of double mutants (aroK aroL) which were shown to be aromatic amino acid auxotrophs (11). The sequence of aroK published by Løbner-Olesen and Marinus (11) described a protein of either 139 or 144 amino acids, depending on the start point of translation, and showing 34% homology with the AroL protein over a limited sequence of 97 amino acids. The published sequence for the aroL-encoded protein describes a protein of 174 amino acids (2).During some studies of a putative aroK mutant (JP1084), we had occasion to sequence the aroK gene in this strain and in a related aroK ϩ strain (JP7615). We discovered a number of errors in the published sequence of the aroK region (10, 11, 13) which, when corrected, alter the length of the aroK coding region to 173 amino acids and extend the homology between the two shikimate kinase enzymes to their entire length. Since we observed no differences in the aroK sequences from JP7615 and JP1084, we conclude that JP1084 is not mutated in aroK and probably carries a mutation in aroB which is in the same transcriptional unit as aroK (10).Cloning and sequencing aroK. Chromosomal DNA was obtained from strains JP7615 and JP1084 by the method of Slauch and Silhavy (21) and used as the DNA template in PCRs. The DNA sequence of Løbner-Olesen et al. (10) was used to design oligonucleotides flanking the aroK coding sequence. The forward primer used in these reactions corresponded to nucleotides (nt) 1443 to 1460 and contained an EcoRI linker at the 5Ј end. The reverse primer corresponded to nt 2275 to 2257 and contained a BamHI linker at the 5Ј end. The DNA fragments were cloned into M13 vectors, and both strands were sequenced from three independent clones (20). Compressions were resolved by replacing dGTP with dITP in the sequencing reactions by the method of Mills and Kramer (15). When the sequence analysis indicated that the aroK reading frame e...