Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12.

We used site-directed mutagenesis to replace the Escherichia coli tryptophanase (tna) operon leader peptide start codon with AUC. This change greatly decreased the uninduced rate of tna operon expression, and it also lowered the response to inducer. We conclude that leader peptide synthesis plays an essential role in tna operon expression.     

Translation of the leader region of the Escherichia coli tryptophan operon.

When the trp operon of Escherichia coli contains either of two deletions that fuse the initial portion of the leader region to the distal segments of the trpE gene, novel fusion polypeptides are produced. The new polypeptides are synthesized efficiently both in vivo and in vitro, and their synthesis is subject to repression by trp repressor. Fingerprint analyses of tryptic and chymotryptic digests of the new polypeptides show that both contain trpE polypeptide sequences and, despite their different sizes, share the same N-terminal sequence. Our results suggest that synthesis of the new polypeptides is initiated at the AUG-centered ribosome-binding site in the leader region and proceeds in phase to the region coding for the C-terminal end of the trpE polypeptide.     

Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.

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 eliminate 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 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.     

Use of the tna operon as a new molecular target for Escherichia coli detection

A quantitative real-time PCR targeting the tnaA gene was studied to detect Escherichia coli and distinguish E. coli from Shigella spp. These microorganisms revealed high similarity in the molecular organization of the tna operon.     

Escherichia coli tryptophan operon directs the in vivo synthesis of a leader peptide.

Here we report the identification of the Escherichia coli trp leader peptide synthesized in vivo. We identified the peptide in UV-irradiated maxicells by selective labeling with radioactive amino acids which are included in the predicted sequence of this peptide. Our results support the hypothesis that translation of the peptide-coding region of the leader RNA has a role in the mechanism of attenuation of biosynthetic operons in general and in the E. coli trp operon in particular.     

Dual regulation by arginine of the expression of the Escherichia coli argECBH operon.

The correlation between the level of messenger ribonucleic acid (mRNA) specific for the argECBH gene cluster (argECBH mRNA) measured by ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization and the rates of synthesis of N-acetylornithine deacetylase (argE enzyme) and of argininosuccinate lyase (argH enzyme) of Escherichia coli strain K-12 were determined for steady-state growth with and without added L-arginine and during the transition periods between these two states. During the transient period after arginine removal (transient derepression), the synthesis of enzymes argE and argH was initially three to five times greater than the steady-state derepressed rate finally reached 50 min later. The level of argECHB mRNA correlated well both quantitatively and temporally with the rates of enzyme synthesis during this transition. The level of in vivo charged arginyl-transfer RNA (tRNAarg), monitored simultaneously, was initially only 5 to 10% and gradually increased to a final level of 80% after 45 min. During the transient period after arginine addition (transient repression), the rates of synthesis of enzymes argE and argH decreased to almost zero and gradually reached steady-state repressed rates after about 180 min. The argECBH mRNA level remained constant at the steady-state repressed level throughout transient repression, revealing a discontinuity between the level of this mRNA and rates of enzyme synthesis. A similar discrepancy was noted during the transition after ornithine addition. In vivo charged tRNAarg remained constant at 80% during this transition. After removal of arginine, the zero-level transient enzyme synthesis developed after only 7.5 min of arginine deprivation and was maximum after 30 min. The results suggest an accumulation of a molecule regulated by arginine that plays a role in transient repression. Our data indicate that arginyl-tRNA synthetase is not this molecule since its synthesis was unaffected by arginine. The ratios of steady-state argE and argH enzyme synthesis without arginine to that with arginine were 12 and 20, respectively, whereas the similar ratio for argECBH mRNA was 2 to 3. The repressed level of argECBH mRNA was not affected by attempts to repress or derepress the ppc+ gene (carried on the DNA used for hybridization), and the repressed level of argECBH mRNA was lowered about 50% in cells carrying an internal argBH deletion. These data taken together indicate the presence of an excess of untranslated argECBH mRNA during both transient and steady-state repression by arginine. Thus, a second regulatory mechanism, not yet defined, appears to play an important role in arginine regulation of enzyme synthesis.     

Stereochemistry and mechanism of reactions catalyzed by tryptophanase Escherichia coli.

Several beta replacement and alpha,beta elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)- and (2s,3s)-[3(-3H)]serine in the presence of indole, deamination of these serines in D2O to pyruvate and ammonia, and cleavage of (2S,3R)-and (2S,3S)-[3(-3H)]tryptophan in D2O to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,3H]pryuvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer of the alpha proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactor.substrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff's base. Degradation of the resulting pyridoxylalanine to (2S)-[2(-3H)]alanine and (4'S)-[4'(-3H)]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4' of the complex and that the ketimine double bond is trans.     

Essential arginine residues in tryptophanase from Escherichia coli.

Tryptophanase from Escherichia coli B/1t7-A is inactivated by the arginine-specific reagent, phenylglyoxal, in potassium phosphate buffer at pH 7.8 AND 25 degrees. Apo- and holoenzyme are inactivated at the same rate, and inactivation of both is correlated with modification of 2 arginine residues/tryptophanase monomer. Substrate analogs having a carboxyl group protect the holoenzyme against both inactivation and arginine modification but have no effect on the inactivation or modification of the apoenzyme. Phenylglyoxal-modified apotryptophanase retains the capacity to bind the coenzyme, pyridoxal-P, but the spectrum of this reconstituted species differs from that of native holotryptophanase. Neither this reconstituted species nor the phenyglyoxal-modified holoenzyme shows the 500 nm absorption characteristic of the native enzyme when substrates are added. These results demonstrate a requirement for specific arginine residues for substrate binding and are discussed in the context of the known conformational and spectal forms of tryptophanase with regard to a possible role for arginine residues in formation of a catalytically effective enzyme-pyridoxal-P complex.