Regulation of the Escherichia coli tryptophan operon by early reactions in the aromatic pathway

7-Methyltryptophan (7MT) or compounds which can be metabolized to 7MT, 3-methylanthranilic acid (3MA) and 7-methylindole, cause derepression of the trp operon through feedback inhibition of anthranilate synthetase. Tyrosine reverses 3MA or 7-methylindole derepression, apparently by increasing the amount of chorismic acid available to the tryptophan pathway. A mutant isolated on the basis of 3MA resistance (MAR 13) was found to excrete small amounts of chorismic acid and to have a feedback-resistant phenylalanine 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. Genetic evidence indicates that the mutation conferring 3MA resistance and feedback resistance is very closely linked to aroG, the structural gene for the DAHP synthetase (phe). Since feedback inhibition of anthranilate synthetase by l-tryptophan (or 7MT) is competitive with chorismic acid, alterations in growth conditions (added tyrosine) or in a mutant (MAR 13) which increase the amount of chorismic acid available to the tryptophan pathway result in resistance to 7MT derepression. Owing to this competitive nature of tryptophan feedback inhibition of anthranilate synthetase by chorismic acid, the early pathway apparently serves to exert a regulatory influence on tryptophan biosynthesis.     

Dynamic diversity and small-molecule evolution: a new paradigm for ligand identification.

A longstanding goal of organic, medicinal and bioorganic chemists has been the discovery of efficient methods for designing or identifying biologically active compounds. Recently, several groups have reported using the directed evolution of combinatorial libraries as a new method of identifying compounds capable of binding tightly to a target molecule. Although significant development remains to be done, the initial results suggest that dynamic diversity and associated selection methods will prove to be valuable additions to the drug-discovery process.     

Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase

While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria. Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria. Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway. This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans. Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R. metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU). Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.     

The tryptophan pathway genes of the Sargasso Sea metagenome: new operon structures and the prevalence of non-operon organization

Background  

The enormous database of microbial DNA generated from the Sargasso Sea metagenome provides a unique opportunity to locate genes participating in different biosynthetic pathways and to attempt to understand the relationship and evolution of those genes. In this article, an analysis of the Sargasso Sea metagenome is made with respect to the seven genes of the tryptophan pathway.     

Experimental evolution of a novel pathway for glycerol dissimilation inEscherichia coli

Wild-type Escherichia coli utilizes glycerol aerobically through an inducible pathway mediated by an ATP-dependent kinase and a glycerol 3-phosphate dehydrogenase which is a flavoprotein. A mutant, strain ECL424, employing a novel pathway for glycerol utilization was isolated. The novel pathway is mediated by an NAD-linked dehydrogenase and a dihydroxyacetone specific enzyme II of the phosphoenolpyruvate phosphotransferase system. This study describes the selection from strain ECL424, a derivative which grows more rapidly on glycerol. The derivative, strain ECL428, produces twice the parental levels of both the dehydrogenase and the enzyme II during growth on glycerol. The function of the dehydrogenase in wild-type cells is unknown, although hydroxyacetone (acetol), 3-hydroxy-2-butanone (acetoin), and 1-amino-2-propanone are gratuitous inducers. The induction can be prevented by glucose whose effect can be cancelled by external cyclic AMP. The effects of hydroxyacetone, glucose, and cyclic AMP are attenuated in the two mutants in which the dehydrogenase is produced at high basal levels. The dihydroxyacetone specific enzyme II is inducible by the substrate in both wild-type and mutant strains and serves for growth on the triose.     

Modeling operon dynamics: the tryptophan and lactose operons as paradigms

Understanding the regulation of gene control networks and their ensuing dynamics will be a critical component in the understanding of the mountain of genomic data being currently collected. This paper reviews recent mathematical modeling work on the tryptophan and lactose operons which are, respectively, the classical paradigms for repressible and inducible operons.     

Temperature-sensitive repression of the tryptophan operon in Escherichia coli

Mutants of Escherichia coli exhibiting temperature-sensitive repression of the tryptophan operon have been isolated among the revertants of a tryptophan auxotroph, trpS5, that produces an altered tryptophanyl transfer ribonucleic acid (tRNA) synthetase. Unlike the parental strain, these mutants grew in the absence of tryptophan at high but not at low temperature. When grown at 43.5 C with excess tryptophan (repression conditions), they produced 10 times more anthranilate synthetase than when grown at 36 C or lower temperatures. Similar, though less striking, temperature-sensitivity was observed with respect to the formation of tryptophan synthetase. Transduction mapping by phage P1 revealed that these mutants carry a mutation cotransducible with thr at 60 to 80%, in addition to trpS5, and that the former mutation is primarily responsible for the temperature-sensitive repression. These results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon. In agreement with this conclusion, tRNA of several trpR mutants was found to be normal with respect to its tryptophan acceptability. It was also shown that the trpS5 allele, whether present in trpR or trpR(+) strains, produced appreciably higher amounts of anthranilate synthetase than the corresponding trpS(+) strains under repression conditions. This was particularly true at higher temperatures. These results provide further evidence for our previous conclusion that tryptophanyl-tRNA synthetase is somehow involved in repression of this operon.     

Theoretical study of tryptophan operon: Application in microbial technology

A model for the tryptophan operon is formulated based on the genetic and biophysical data available on the structure of the operon and the nature of interactions between the represser and its ligands. Studies have been done, on wild-type, superrepressing, and loose-binding strains to identify conditions at which the stability of the system changes (i.e., evolves to a stable synthesis or periodic synthesis with increasing amplitude). Also, the factors that increase the yield of tryptophan are studied and predictions made, based on the results, for obtaining overproducing strains of tryptophan that can be used for the industrial production of this useful amino acid.