Analysis in vivo of factors affecting the control of transcription initiation at promoters containing target sites for trp repressor

An investigation of repression in the trp system of Escherichia coli was undertaken using operon fusions and plasmids constructed via recombinant DNA technology. The promoters of the trp operon and the trpR gene were fused to lacZ, enabling the activity of these promoters to be evaluated under various conditions through measurements of beta-galactosidase production. In confirmation of earlier studies, the trpR gene was shown to be regulated autogenously. This control feature of the trp system was found to maintain intracellular Trp repressor protein at essentially invariant levels under most conditions studied. Increasing the trpR+ gene dosage did not significantly elevate Trp repressor protein levels, nor did the introduction of additional operator "sinks" result in significantly decreased levels of Trp repressor protein. Definite alterations in intracellular Trp repressor protein levels were achieved only by subverting the normal trpR regulatory elements. The placement of the lacUV5 or the lambda PL promoters upstream of the trpR gene resulted in significant increases in repression of the trp system. Substituting the primary trp promoter/operator for the native trpR promoter/operator resulted in an altered regulatory response of the trp system to tryptophan limitation or excess. The regulation of the trpR gene effectively imparts a broad range of expression to the trp operon in a manner finely attuned to fluctuations in intracellular tryptophan levels.     

Tryptophan biosynthesis in Salmonella typhimurium: location in trpB of a genetic difference between strains LT2 and LT7.

Salmonella typhimurium prototrophs carrying a trpR mutation synthesize tryptophan biosynthetic enzymes constitutively. When feedback inhibition of anthranilate synthetase but not 5'-phosphoribosylpyrophosphate phosphoribosyltransferase activity was by-passed by growing cells on media supplemented with anthranilic acid, all trpR prototrophs overproduced and excreted tryptophan. However, the rate of tryptophan production depended on both the ancestry of the trpR strain and the integrity of its trpA gene. Prototrophs with trp genes derived from S. typhimurium strain LT2 produced tryptophan more efficiently than those with trp genes derived from strain LT7. This strain difference was cryptic insofar as it did not affect the growth rate; it was revealed only as a rate-limiting step in the constitutive biosynthesis of tryptophan in the presence of anthranilic acid, and was due to a lesion in the LT7-derived trpB gene. Strains with LT7-derived trp genes bearing a deletion in trpA produced tryptophan as readily as LT2 trpR prototrophs. This indicated that LT7-specific 5-phosphoribosylpyrophosphate phosphoribosyltransferase must be aggregated with the trpA gene produce to give an observable reduction of constitutive tryptophan production. The discovery of this strain difference has particular implications for studies involving the activities of trpA and B genes and their products in S. typhimurium and may have general significance for other studies involving different strains of Salmonella.     

Effect of tryptophan analogs on derepression of the Escherichia coli tryptophan operon by indole-3-propionic acid.

The abilities of 14 tryptophan analogs to repress the tryptophan (trp) operon have been studied in Escherichia coli cells derepressed by incubation with 0.25 mM indole-3-propionic acid (IPA). trp operon expression was monitored by measuring the specific activities of anthranilate synthase (EC 4.1.3.27) and the tryptophan synthase (EC 4.2.1.20) beta subunit. Analogs characterized by modification or removal of the alpha-amino group or the alpha-carboxyl group did not repress the trp operon. The only analogs among this group that appeared to interact with the trp aporepressor were IPA, which derepressed the trp operon, and d-tryptophan. Analogs with modifications of the indole ring repressed the trp operon to various degrees. 7-Methyl-tryptophan inhibited anthranilate synthase activity and consequently derepressed the trp operon. Additionally, 7-methyltryptophan prevented IPA-mediated derepression but, unlike tryptophan, did so in a non-coordinate manner, with the later enzymes of the operon being relatively more repressed than the early enzymes. The effect of 7-methyltryptophan on IPA-mediated derepression was likely not due to the interaction of IPA with the allosteric site of anthranilate synthase, even though feedback-resistant mutants of anthranilate synthase were partially resistant to derepression by IPA. The effect of 7-methyltryptophan on derepression by IPA was probably due to the effect of the analog-aporepressor complex on trp operon expression.     

Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli

A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.     

New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.

For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established.     

Tryptophan operon regulation in interspecific hybrids of enteric bacteria.

We examined tryptophan regulation in merodiploid hybrids in which a plasmid carrying the trp operon of Escherichia was introduced into Trp mutants of other enteric genera, or in which a plasmid carrying the trpR+ (repressor) gene of E. coli was transfered into fully constitutive trpR mutants of other genera. In these hybrids the trp operon of one species is controlled by the repressor of a different species. Similar investigations were possible in transduction hybrids in which either the trp operon or the trpR+ locus of Shigella dysenteriae was introduced into E. coli. Our measurements of trp enzymes levels in repressed and nonrepressed cells indicate that Trp regulation is normal, with only minor quantitative variations, in hybrids between E coli and Shigella dysenteriae, Salmonella typhimurium, Klebsiella aerogenes, Serratia marcescens, and Proteus mirabilis. Our results support the idea that a repressor-operator mechanism for regulating trp messenger ribonucleic acid production evolved in a common ancestor of the enteric bacteria, and that this repressor-operator recognition has been conversed during the evolutionary divergence of the Enterobacteriaceae.     

Genetic control of tryptophan hypersynthesis in regulatory mutants of Pseudomonas putida

The 6-fluorotryptophan resistant MR1 mutant was obtained from Pseudomonas putida M30 (Tyr- Phe-) strain. The mutant was able to excrete tryptophan (60 micrograms/ml) and has derepressed aroF gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase. The mutation isolated was identified as aroR with the help of cloning early aroF gene of P. putida. On the next step of selection, regulatory mutant MR2 was obtained producing 240 micrograms/ml of tryptophan. The MR2 has derepressed unlinked trpE and trpDC genes and represents a mutant of the trpR type. Expression of the trpE gene of P. putida MR2 weakened in the presence of tryptophan excess in the medium, which points to attenuation of this gene. From the prototrophic variant of P. putida MR2 the MRP3 mutant producing 850 micrograms/ml of tryptophan was obtained. This mutant was characterized by twofold increase in the activity of the anthranilate synthase encoded by the trpE gene. The assay of the activity of tryptophanyl-tRNA synthase in P. putida MRP3 demonstrated that the mutant has TrpS+ phenotype.     

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.     

Location of trpR Mutations in the serB-thr Region of Salmonella typhimurium

Tryptophan biosynthesis in Salmonella is controlled by at least one regulatory gene, trpR, which is cotransducible with thr genes and not with the trp operon. Mutations in trpR cause derepression of tryptophan enzyme synthesis and confer resistance to growth inhibition by 5-methyltryptophan. Nineteen trpR mutations were mapped with respect to thrA and serB markers by two-point (ratio) and three-point transduction tests. The results are all consistent with the site order serB80-trpR-thrA59 on the Salmonella chromosome. Very low or undetectable levels of recombination between different trpR mutations have so far prevented the determination of fine structure in the trpR gene. Thirteen other 5-methyltryptophan-resistant mutants previously found not to be cotransducible with either the trp operon or thrA, and designated trpT, were also used in these experiments. Lack of cotransducibility with thrA was confirmed, and no linkage with serB was detected. The nature and location of trpT mutations remain obscure.     

Isolation of auxotrophs and analysis of regulation of tryptophan biosynthesis in Zymomonas mobilis

Tryptophan auxotrophs were isolated and used to analyze the regulation of tryptophan biosynthesis in Zymomonas mobilis. Twelve tryptophan auxotrophs were cassified as trp E, B or A based on accumulation of, or growth on, indole and anthranilic acid. Trp B mutants were found to accumulate indole when grown on limiting, but not on excess tryptophan, suggesting that tryptophan plays a role in regulating its biosynthesis. Tryptophan synthase and indoleglycerol phosphate synthase specific activities were measured in the wild-type strain and two trp mutants grown in limiting or excess tryptophan. Neither activity was repressed by exogenous tryptophan.Abbreviations CDRP O-(carboxyphenol amino)-1 deoxyribulose 5-phosphate - IGPS indoleglycerol phosphate synthase - TS tryptophan synthase Dedicated in memory of Dr. O. H. Smith     

Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit.

Suppressors of the blue fluorescence phenotype of the Arabidopsis trp1-100 mutant can be used to identify mutations in genes involved in plant tryptophan biosynthesis. Two recessive suppressor mutations define a new gene, TRP4. The trp4 mutant and the trp1-100 mutant are morphologically normal and grow without tryptophan, whereas the trp4; trp1-100 double mutant requires tryptophan for growth. The trp4; trp1-100 double mutant does not segregate at expected frequencies in genetic crosses because of a female-specific defect in transmission of the double mutant genotype, suggesting a role for the tryptophan pathway in female gametophyte development. Genetic and biochemical evidence shows that trp4 mutants are defective in a gene encoding the beta subunit of anthranilate synthase (AS). Arabidopsis AS beta subunit genes were isolated by complementation of an Escherichia coli anthranilate synthase mutation. The trp4 mutation cosegregates with one of the genes, ASB1, located on chromosome 1. Sequence analysis of the ASB1 gene from trp4-1 and trp4-2 plants revealed different single base pair substitutions relative to the wild type. Anthranilate synthase alpha and beta subunit genes are regulated coordinately in response to bacterial pathogen infiltration.     

Action of tryptophan analogues in Saccharomyces cerevisiae

In an analysis of the effects of various tryptophan and indole analogues in Saccharomyces cerevisiae we determined the mechanisms by which they cause growth inhibition: 4-Methyltryptophan causes a reduction in protein synthesis and a derepression of the tryptophan enzymes despite of the presence of high internal levels of tryptophan. This inhibition can only be observed in a mutant with increased permeability to the analogue. These results are consistent with but do not prove an interference of this analogue with the charging of tryptophan onto tRNA. 5-Methyltryptophan causes false feedback inhibition of anthranilate synthase, the first enzyme of the tryptophan pathway. This inhibits the further synthesis of tryptophan and results in results in tryptophan limitation, growth inhibition and derepression of the enzymes. Derepression eventually allows wild type cells to partially overcome the inhibitory effect of the analogue. 5-Fluoroindole is converted endogenously to 5-fluorotryptophan by tryptophan synthase. Both endogenous and externally supplied 5-fluorotryptophan are incorporated into protein. This leads to intoxication of the cells due to the accumulation of faulty proteins. 5-Fluorotryptophan also causes feedback inhibition of anthranilate synthase and reduces the synthesis of tryptophan which would otherwise compete with the analogues in the charging reaction. Indole acrylic acid inhibits the conversion of indole to tryptophan by tryptophan synthase. This results in a depletion of the tryptophan pool which, in turn, causes growth inhibition and derepression of the tryptophan enzymes.Abbreviations cpm counts per minute - OD optical density at 546 nm - TCA trichloro acetic acid - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for the corresponding tryptophan biosynthetic enzymes - trpl^– res. trp1^± refer to mutant strains synthesizing completely resp. partially defective enzymes     

Frameshifting in the expression of the Escherichia coli trpR gene

The trpR gene of Escherichia coli carries an open reading frame that encodes the trp repressor, 108 amino acids long. Here we show that translation of an additional (+1) reading frame of trpR occurs both in vivo and in vitro. This results in the synthesis of a stable +1 frame polypeptide. Using site-specific mutagenesis, immunological techniques and amino acid sequencing we have found that the N-terminus of the +1 frame product and that of the known 0 frame product are identical but that their C-termini differ. Our results are discussed in relation to the role of natural frameshifting as a regulatory mechanism of gene expression in general, and with respect to tryptophan biosynthesis in particular.     

Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.     

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation.     

Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase beta genes.

The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.