Escherichia coli forms three permeases that can transport the amino acid tryptophan: Mtr, AroP, and TnaB. The structural genes for these permeases reside in separate operons that are subject to different mechanisms of regulation. We have exploited the fact that the tryptophanase (tna) operon is induced by tryptophan to infer how tryptophan transport is influenced by the growth medium and by mutations that inactivate each of the permease proteins. In an acid-hydrolyzed casein medium, high levels of tryptophan are ordinarily required to obtain maximum tna operon induction. High levels are necessary because much of the added tryptophan is degraded by tryptophanase. An alternate inducer that is poorly cleaved by tryptophanase, 1-methyltryptophan, induces efficiently at low concentrations in both tna+ strains and tna mutants. In an acid-hydrolyzed casein medium, the TnaB permease is most critical for tryptophan uptake; i.e., only mutations in tnaB reduce tryptophanase induction. However, when 1-methyltryptophan replaces tryptophan as the inducer in this medium, mutations in both mtr and tnaB are required to prevent maximum induction. In this medium, AroP does not contribute to tryptophan uptake. However, in a medium lacking phenylalanine and tyrosine the AroP permease is active in tryptophan transport; under these conditions it is necessary to inactivate the three permeases to eiiminate tna operon induction. The Mtr permease is principally responsible for transporting indole, the degradation product of tryptophan produced by tryptophanase action. The TnaB permease is essential for growth on tryptophan as the sole carbon source. When cells with high levels of tryptophanase are transferred to a tryptophan-free growth medium, the expression of the tryptophan (trp) operon is elevated. This observation suggests that the tryptophanase present in these cells degrades some of the synthesized tryptophan, thereby creating a mild tryptophan deficiency. Our studies assign roles to the three permeases in tryptophan transport under different physiological conditions. Escherichia coli uses several mechanisms to regulate the expression of its tryptophan (trp) operon and control the rate of tryptophan biosynthesis. The most important of these are repression, transcription attenuation, and feedback inhibition (29). Their combined action permits the bacterium to vary the rate of tryptophan production over a several thousand-fold range. Since tryptophan is costly to produce, efficient shutdown of synthesis is advantageous to the bacterium whenever the amino acid is present in its environment. Consistent with this conclusion, most organisms that feed on other organisms have lost the capacity to synthesize tryptophan.

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Studies of tryptophan synthetase A-protein mutants have shown a structural relationship between the positions of amino acid changes associated with forward mutations and second-site reverse mutations.

Physiological studies were performed under nutritional stress and nonstress conditions to assess the relative importance of the various regulatory mechanisms that Escherichia coli can use to alter its rate of tryptophan synthesis. Mutants were examined in which the tip repressor was inactive, transcription termination at the trp attenuator was altered, transcription initiation at the tip promoter was reduced, or feedback inhibition of anthranilate synthase was abolished. Strains were examined in media with and without tryptophan, phenylalanine and tyrosine, or acid-hydrolyzed casein and following shifts from one medium to another. Growth rates and anthranilate synthase levels were measured. In media lacking tryptophan, each of the mutants showed relief of repression and/or attenuation and maintained a near-normal growth rate. Following a shift from a medium containing tryptophan to a trptophan-free medium containing phenylalanine and tyrosine or acid-hydrolyzed casein, mutants with abnormally low trp enzyme levels exhibited an appreciable growth lag before resuming growth. The wild-type strain displayed termination relief only under one extreme shift condition, upon transfer from a minimal medium containing tryptophan to minimal medium with only phenylalanine and tyrosine. A promoter down-mutant had difficulty adjusting to a shift from high tryptophan to low tryptophan levels in a medium containing acid-hydrolyzed casein. In all media tested, anthranilate synthase levels were lower in a feedback-resistant mutant than in the wild type. These studies demonstrate the capacity of E. coli to adjust its rate of tryptophan synthesis to maintain rapid growth following a shift to stressful nutritional conditions. Escherichia coli has the ability to adjust its rate of tryptophan biosynthesis over a several-thousand-fold range. This impressive regulatory capacity emanates from the use of a relatively strong promoter to drive tip operon expression, repression and attenuation to regulate tip operon transcription, and feedback inhibition to regulate entry of the common aromatic precursor, chorismate, into the tryptophan biosynthetic pathway (36). In addition, tryptophan influences the flow of carbon into and through the common aromatic pathway that culminates in chorismate production (23 (415) 725-8221. tion at the attenuator. Attenuation regulates transcription of the structural genes of the tip operon ca. six-to eightfold (11, 13). There is one additional regulatory feature of transcriptional attenuation in the tip operon. Cells that are incapable of initiating synthesis of the tip leader peptide experience five times greater termination at the attenuator than cells that can perform this function (24,33,43). This phenomenon has been termed superattenuation (33). Events that prevent or decrease synthesis of the tip leader peptide could cause superattenuation and lead to an additional fivefold increase in termination at the attenuator. Feedback inhibition of anthranilate synthase (ASase), the enzyme catalyzing the first r...