Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.     

Repression of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (trp) and enzymes of the tryptophan pathway in Escherichia coli K-12

Mutant strains of Escherichia coli K-12 have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (trp) is partially constitutive. The mutation causing derepression is closely linked to aroH [the structural gene for DAHP synthetase (trp)] and occurs in a locus designated aroJ. The aroJ mutation is not recessive in an aroJ(+)/aroJ(-) diploid strain, as the synthesis of DAHP synthetase (trp) is still derepressed in this strain. On the basis of its close linkage to aroH and its continued expression in an aroJ(+)/aroJ(-) diploid, it is postulated that aroJ is an operator locus controlling the expression of the structural gene aroH. In support of this conclusion, the synthesis of anthranilate synthetase is still normally repressible in aroJ(-) strains, whereas, in trpR(-) strains, both DAHP synthetase (trp) and anthranilate synthetase are synthesized constitutively. The synthesis of DAHP synthetase (trp) remains repressible in an operator-constitutive mutant of the tryptophan operon. In two trpS mutants which possess defective tryptophanyl transfer ribonucleic acid synthetase enzymes, neither DAHP synthetase (trp) nor anthranilate synthetase derepress under conditions in which the defective synthetase causes a decrease in growth rate. On the other hand, an effect of the trpS mutant alleles on the level of anthranilate synthetase has been observed in strains which are derepressed for the synthesis of this enzyme, because of a mutation in the gene trpR. Possible explanations for this effect are presented.     

Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans.

Sixty independent tryptophan auxotrophs of Pseudomonas acidovorans were isolated and characterized for nutritional response to intermediates of the pathway, accumulation of intermediates, and levels of tryptophan-synthetic enzymes. Mutants for each of the seven proteins catalyzing the five steps of tryptophan synthesis were obtained. Transductional analysis established three unlinked chromosomal regions: trpE, trpGDC, and trpFBA. The order of the genes within the two clusters was not determined. The levels and enzymatic activities of wild-type and mutant strains indicated that trpE and trpGDC were repressed by tryptophan. In contrast, trpFBA was not derepressed significantly by starvation for tryptophan. The trpG mutants had an additional requirement for p-aminobenzoate, which suggested that anthranilate synthase subunit II also served as glutamine-binding protein in the analogous reaction catalyzed by p-aminobenzoate synthase. In addition, trpD mutants revealed the ability of P. acidovorans to degrade anthranilate via the beta-ketoadipate pathway.     

An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene.

McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.     

Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase.

Biochemical and immunological experiments have suggested that the Escherichia coli enzyme p-aminobenzoate synthetase and anthranilate synthetase are structurally related. Both enzymes are composed of two nonidentical subunits. Anthranilate synthetase is composed of proteins encoded by the genes trp(G)D and trpE, whereas p-aminobenzoate synthetase is composed of proteins encoded by pabA and pabB. These two enzymes catalyze similar reactions and produce similar products. The nucleotide sequences of pabA and trp(G)D have been determined and indicate a common evolutionary origin of these two genes. Here we present the nucleotide sequence of pabB and compare it with that of trpE. Similarities are 26% at the amino acid level and 40% at the nucleotide level. We propose that pabB and trpE arose from a common ancestor and hence that there is a common ancestry of genes encoding p-aminobenzoate synthetase and anthranilate synthetase.     

Mutant Strains of Escherichia coli K-12 Exhibiting Enhanced Sensitivity to 5-Methyltryptophan

Eighteen mutants (designated MT(s)), isolated in Escherichia coli K-12, showed increased sensitivity to inhibition of growth by 5-methyltryptophan. All mutants were also much more sensitive to 4-methyltryptophan and 7-azatryptophan but exhibited near normal sensitivity to 5-fluorotryptophan and 6-fluorotryptophan. All of the mutations were linked to the trp operon. Their locations within the trp operon were established by deletion mapping. There was good agreement between the map position of an MT(s) mutation and a lowered activity of one of the tryptophan pathway enzymes. Three mutants, one of which contained a mutation that mapped within the trpE gene, were deficient in their ability to use glutamine as an amino donor in the formation of anthranilic acid. Another trpE mutation led to the production of an anthranilate synthetase with an increased sensitivity to feedback inhibition by tryptophan.     

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.     

Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase

An improved method was developed to align related protein sequences and search for homology. A glutamine amide transfer domain was identified in an NH2-terminal segment of GMP synthetase from Escherichia coli. Amino acid residues 1-198 in GMP synthetase are homologous with the glutamine amide transfer domain in trpG X D-encoded anthranilate synthase component II-anthranilate phosphoribosyltransferase and the related pabA-encoded p-aminobenzoate synthase component II. This result supports a model for gene fusion in which a trpG-related glutamine amide transfer domain was recruited to augment the function of a primitive NH3-dependent GMP synthetase. Sequence analyses emphasize that glutamine amide transfer domains are thus far found only at the NH2 terminus of fused proteins. Two rules are formulated to explain trpG and trpG-related fusions. (i) trpG and trpG-related genes must have translocated immediately up-stream of genes destined for fusion in order to position a glutamine amide transfer domain at the NH2 terminus after fusion. (ii) trpG and trpG-related genes could not translocate adjacent to a regulatory region at the 5' end of an operon. These rules explain known trpG-like fusions and explain why trpG and pabA are not fused to trpE and pabB, respectively. Alignment searches of GMP synthetase with two other enzymes that bind GMP, E. coli amidophosphoribosyltransferase and human hypoxanthine-guanine phosphoribosyltransferase, suggest a structurally homologous segment which may constitute a GMP binding site.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.     

Naturally occurring sites within the Shigella dysenteriae tryptophan operon severely limit tryptophan biosynthesis.

We investigated the structural, functional, and regulatory properties of the Shigella dysenteriae tryptophan (trp.) operon in transduction hybrids in which the cysB-trp-region of Escherichia coli is replaced by the corresponding region from S. dysenteriae. Tryptophan biosynthesis was largely blocked in the hybrids, although the order of the structural genes was identical with that of E. coli. Nutritional tests and enzyme assays revealed that the hybrids produced a defective anthranilate synthetase (ASase). Deletion mapping identified two distinct sites in trpE, each of which was partially responsible for the instability and low activity of ASase. We also discovered a pleiotropic site trpP (S) that maps outside the structural gene region and is closely linked to the S. dysenteriae trp operator. trpP (S) reduced the rate of trp messenger ribonucleic acid synthesis, and consequently trp enzyme levels, 10-fold relative to wild-type E. coli. In recombinants in which the structural genes of E coli were under the control of the S. dysenteriae promoter, enzyme levels were also reduced 10-fold. In some fast-growing revertants of the original hybrids, the rates of trp messenger ribonucleic acid synthesis and levels of tryptophan synthetase were restored to values characteristic of wild-type E.coli. Thus, the Trp auxotrophy associated with the S dysenteriae trp operon derives from the combination of a defective ASase and decreased expression of the entire operon imposed by trpP (S).     

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.     

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 Analog Resistance Mutations in Chlamydomonas Reinhardtii

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.     

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.     

Rhizobium meliloti mutants unable to synthesize anthranilate display a novel symbiotic phenotype.

Analyses of Rhizobium meliloti trp auxotrophs suggest that anthranilate biosynthesis by the R. meliloti trpE(G) gene product is necessary during nodule development for establishment of an effective symbiosis. trpE(G) mutants, as well as mutants blocked earlier along this pathway in aromatic amino acid biosynthesis, form nodules on alfalfa that have novel defects. In contrast, R. meliloti trp mutants blocked later in the tryptophan-biosynthetic pathway form normal, pink, nitrogen-fixing nodules. trpE(G) mutants form two types of elongated, defective nodules containing unusually extended invasion zones on alfalfa. One type contains bacteroids in its base and is capable of nitrogen fixation, while the other lacks bacteroids and cannot fix nitrogen. The trpE(G) gene is expressed in normal nodules. Models are discussed to account for these observations, including one in which anthranilate is postulated to act as an in planta siderophore.