Restoration of Phosphoribosyl Transferase Activity by Partially Deleting the trpB Gene in the Tryptophan Operon of Salmonella typhimurium

The amber mutant trpA28, which contains a mutation mapping within the so-called "unusual" region of the tryptophan (trp) operon of Salmonella typhimurium (between the genes trpA and trpB), lacks both components of the anthranilate synthetase (AS)-phosphoribosyl transferase (PRT) enzyme complex, the products of the genes trpA and trpB, respectively. Twenty-six revertants of this mutant selected on minimal medium supplemented with anthranilic acid, a substrate of PRT, contain deletions of various segments of the "unusual" region and make a species of PRT different in every respect from the wild-type, dissociated form of this enzyme. The results indicate that the unusual region corresponds to the operator proximal end of the trpB gene. Mutants in the unusual region, however, show unexpectedly low levels of AS activity and in two cases (trpA515 and trpA28) no detectable activity of this enzyme component.     

Nucleotide sequences and genomic constitution of five tryptophan genes of Lactobacillus casei

Five trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escherichia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6,468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of tryptophan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.     

Polar and Antipolar Mutants in the Tryptophan Operon of Salmonella typhimurium

Polar mutations in trpA, the first structural gene of the tryptophan operon of Salmonella typhimurium, have an uncoordinate effect on the expression of the distal genes, with trpB, the second gene, being more drastically affected than the last three. A number of these polar mutant strains grow very poorly on anthranilic acid-supplemented minimal medium. By selecting for more rapid growth in the presence of anthranilic acid, secondary mutant clones showing a correction of the polar effect were isolated. A few of these were analyzed and shown to contain deletions of various segments of the trpA gene. Ten randomly isolated deletion mutants missing various segments of the trp operon were analyzed for possible pleiotropic effects. Five of them showed a pleiotropic effect of some sort and five did not. Of those showing pleiotropic effects, one had lost the promotor-like elements necessary to initiate expression of the operon, three showed possible antipolar effects, and one showed both polar and antipolar effects simultaneously.     

Analysis of Polar and Nonpolar Tryptophan Mutants by Derepression Kinetics

A comparison of the rates of synthesis of the tryptophan biosynthetic enzymes of Salmonella typhimurium under derepression showed that the genes of the trp operon can be expressed in a coordinate fashion in auxotrophs carrying nonpolar mutations. This coordination disappeared in trpA polar mutants. The loss of coordination affected only trpB, the second gene in the operon, which was always more drastically affected than the three distal genes. Polar mutations in trpA, the first gene of the trp operon, reduced the rates of synthesis of the tryptophan biosynthetic enzymes under conditions of derepression. When these rates were measured and correlated with the map position of each polar mutation, a polarity gradient of decreasing intensity (moving distally from the operator end of the gene) was obtained. Certain mutations ("unusual mutations") mapping at the operator distal end of trpA, and considered by other workers to correspond to the operator proximal end of trpB, were found to be polar. The bearing of our observations on the question of coordinate versus semicoordinate expression of the trp genes and the status of the "unusual mutations" is discussed.     

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.     

A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair

Expression of trpB and trpA of the Escherichia coli tryptophan operon is shown to be "translationally coupled", i.e., efficient translation of the trpA coding region is dependent on prior translation of the trpB coding region and termination of translation at the trpB stop codon. To examine the dependence of trpA expression on the ribosome binding site sequence in the distal segment of trpB, deletions were produced that replaced this trpB sequence. Analysis of trpA expression in these deletion mutants established that the ribosome binding site sequence is required for efficient translation of the trpA segment of trp mRNA. A modest effect of translation over the trpA ribosome binding site on independent initiation at that site was also observed.     

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.     

Tryptophan operon of methylotrophic facultative Pseudomonas sp. M bacteria. I. Isolation and characterization of auxotrophic Trp-mutants

Eighteen auxotropic trp- mutants of the facultative methylotrophic bacteria Pseudomonas sp. M. induced by nitrosoguanidine were characterized. Trp- mutants were tested for a number of biochemical properties: the capacity to grow on tryptophan intermediates, their accumulation in growth medium and activities of key enzymes. The trpE, trpD, trpC, trpF, trpB and trpA mutants were identified. The trpDC121 mutant with a one-point mutation has been obtained. This mutation caused inactivation of two enzymes--anthranilate-5-phosphoribosyl transferase and indole-3-glycerophosphate synthase. Unusual trpA and trpB auxotrophs with TrpAB- phenotype were described. It may be concluded that this type of mutations cause loss of catalytic activity of a subunit of tryptophan synthase as well as its structural modification. As a result, no active tryptophan synthase complex is formed and hence, the activity of the opposite intact subunit is inhibited.     

Differences between the anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases of Salmonella typhimurium strains LT2 and LT7.

The anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases (PRT), coded by the second structural gene (trpB) of the tryptophan (trp) operon in strains LT2 and LT7 of Salmonella typhimurium, differ from each other in a number of parameters. These include the apparent Km values for their substrates anthranilic acid and 5-phosphoribosylpyrophosphate, thermostability, sensitivity to substrate inhibition by anthranilic acid, as well as end-product inhibition by tryptophan and specific activity. The PRT of strain LT7 further differs from that of strain LT2 in that its apparent Km for 5-phosphoribosylpyrophosphate is three to seven times higher when associated with anthranilate synthase in the enzyme complex which catalyses the first two steps of tryptophan biosynthesis than in its free uncomplexed form, which the PRT of strain LT2 shows the same apparent Km for this substrate in both its free and complexed forms. These results confirm and extend the finding of Stuttard (1975) that strains LT2 and LT7 differ genetically form each other at a single site within region II of the trpB gene.     

A novel tryptophan synthase beta-subunit from the hyperthermophile Thermotoga maritima. Quaternary structure, steady-state kinetics, and putative physiological role.

Tryptophan synthase catalyzes the last two steps in the biosynthesis of the amino acid tryptophan. The enzyme is an alpha beta beta alpha complex in mesophilic microorganisms. The alpha-subunit (TrpA) catalyzes the cleavage of indoleglycerol phosphate to glyceraldehyde 3-phosphate and indole, which is channeled to the active site of the associated beta-subunit (TrpB1), where it reacts with serine to yield tryptophan. The TrpA and TrpB1 proteins are encoded by the adjacent trpA and trpB1 genes in the trp operon. The genomes of many hyperthermophilic microorganisms, however, contain an additional trpB2 gene located outside of the trp operon. To reveal the properties and potential physiological role of TrpB2, the trpA, trpB1, and trpB2 genes of Thermotoga maritima were expressed heterologously in Escherichia coli, and the resulting proteins were purified and characterized. TrpA and TrpB1 form the familiar alpha beta beta alpha complex, in which the two different subunits strongly activate each other. In contrast, TrpB2 forms a beta(2)-homodimer that has a high catalytic efficiency k(cat)/K(m)(indole) because of a very low K(m)(indole) but does not bind to TrpA. These results suggest that TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles.     

Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB''-trpA gene pair of the Escherichia coli tryptophan operon.

The trpB and trpA coding regions of the polycistronic trp mRNA of Escherichia coli are separated by overlapping translation stop and start codons. Efficient translation of the trpA coding region is subject to translational coupling, i.e., maximal trpA expression is dependent on prior translation of the trpB coding region. Previous studies demonstrated that the trpA Shine-Dalgarno sequence (within trpB) and/or the location of the trpB stop codon influenced trpA expression. To examine the effect of stop codon location specifically, we constructed plasmids in which different nucleotide sequences preceding the trpA start codon were retained, and only the reading frame was changed. When trpB translation proceeded in the wild type reading frame and terminated at the normal trpB stop codon, trpA polypeptide levels were elevated over the levels observed when translation stopped before or after the natural trpB stop codon. The proximity of the trpB stop codon to the trpA start codon therefore markedly influences trpA expression.     

Duplication-translocations of tryptophan operon genes in Escherichia coli

Mutants of Escherichia coli were selected in which a single mutational event had both relieved the polar effect of an early trpE mutation on trpB and simultaneously released the expression of trpB from tryptophan repression. The frequency at which these mutations appeared was roughly equal to the frequency of point mutations. In each of these mutants, the mutation increased the function of trpB and also increased the activity of some, but not all, of the other four tryptophan operon genes. Genetic analysis showed that the mutations were not located within the trp operon since in each case the parental trp operon could be recovered from the mutants. Each mutant was shown to carry a duplication of a trp operon segment translocated to a new position near the trp operon. Polarity is relieved since the trpB duplication-translocation is not in the same operon as the trpE polar mutation. The duplicated and translocated segments are fused to operons not regulated by tryptophan, so trpB function is no longer subject to tryptophan repression. The properties of the mutants indicate that the length of the duplicated segment and the position to which it is translocated differ in each of the seven mutants studied. The duplications are unstable, but the segregation pattern observed is not consistent with a single crossover model for segregation. That such duplication-translocation events generate a variety of new genetic arrangements at a frequency comparable with point mutations suggests they may play an important role in evolution.     

Clustering of the trp genes in Burkholderia (formerly Pseudomonas) cepacia

Abstract Chromosomal location of trp genes of a strain Burkholderia cepacia (formerly Pseudomonas cepacia ) has been determined by transduction using a generalized transducing phage CP75 and by molecular analysis for a cosmid plasmid clone with trp genes isolated from the genomic gene library of the strain. The trp genes were classified into three linkage groups and they all were closely linked on a short chromosomal region probably in the order ( trpA, trpB, trpF)-(trpC, trpD)-trpE .     

Cloning of the trp gene cluster from a tryptophan-hyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence.

Corynebacterium glutamicum ATCC 21850 produces up to 5 g of extracellular L-tryptophan per liter in broth culture and displays resistance to several synthetic analogs of aromatic amino acids. Here we report the cloning of the tryptophan biosynthesis (trp) gene cluster of this strain on a 14.5-kb BamHI fragment. Subcloning and complementation of Escherichia coli trp auxotrophs revealed that as in Brevibacterium lactofermentum, the C. glutamicum trp genes are clustered in an operon in the order trpE, trpD, trpC, trpB, trpA. The cloned fragment also confers increased resistance to the analogs 5-methyltryptophan and 6-fluorotryptophan on E. coli. The sequence of the ATCC 21850 trpE gene revealed no significant changes when compared to the trpE sequence of a wild-type strain reported previously. However, analysis of the promoter-regulatory region revealed a nonsense (TGG-to-TGA) mutation in the third of three tandem Trp codons present within a trp leader gene. Polymerase chain reaction amplification and sequencing of the corresponding region confirmed the absence of this mutation in the wild-type strain. RNA secondary-structure predictions and sequence similarities to the E. coli trp attenuator suggest that this mutation results in a constitutive antitermination response.     

Tryptophan synthetase alpha(5.7-S): novel molecular species formed within Escherichia coli.

A novel molecular species contributes about 5% of the total tryptophan synthetase of Escherichia coli derepressed for the trp operon enzymes. The new species is identified under conditions in which the dissociation of the two nonidentical subunits of the tryptophan synthetase complex is favored. The new species sediments at 5.7S, catalyzes the conversion of indole-3-glycerol phosphate to indole, and has been designated alpha(5.7-S). Although alpha(5.7-S) is not observed in extracts of trpA or trpB mutant strains deficient in the ability to form tryptophan synthetase alpha or beta2 subunits, respectively, a mixture of the two extracts allows the formation of alpha(5.7-S). Similar results are obtained when a homogeneous alpha protein is mixed with an extract of a trpA mutant strain, suggesting that the interaction of alpha and beta2 proteins is obligatory for alpha(5.7-S) formation. One can obtain a beta2 protein preparation that when mixed with a pure alpha protein gives no alpha(5.7-S). Therefore, the interaction of alpha and beta2 proteins alone is not sufficient for the formation of alpha(5.7-S). When a mixture of alpha and beta2 proteins devoid of alpha(5.7-S) is added to extracts of trp deletion mutants, the novel species can be reconstituted in vitro only when deletions are used that carry at least the operator-proximal part of the trpB gene. Therefore, it is concluded that the alpha(5.7-S) species of tryptophan synthetase results from the interaction of the alpha protein, the beta2 protein, and a third component, beta', specified by the deoxyribonucleic acid defined by the end points of two trp deletion mutants.     

Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation

Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.     

Cosmid cloning of five Zymomonas trp genes by complementation of Escherichia coli and Pseudomonas putida trp mutants.

A library of Zymomonas mobilis genomic DNA was constructed in the broad-host-range cosmid pLAFR1. The library was mobilized into a variety of Escherichia coli and Pseudomonas putida trp mutants by using the helper plasmid pRK2013. Five Z. mobilis trp genes were identified by the ability to complement the trp mutants. The trpF, trpB, and trpA genes were on one cosmid, while the trpD and trpC genes were on two separate cosmids. The organization of the Z. mobilis trp genes seems to be similar to the organization found in Rhizobium spp., Acinetobacter calcoaceticus, and Pseudomonas acidovorans. The trpF, trpB, and trpA genes appeared to be linked, but they were not closely associated with trpD or trpC genes.