Conditionally Expressed Missense Mutations: The Basis for the Unusual Phenotype of an Apparent trpD Nonsense Mutant of SALMONELLA TYPHIMURIUM

Tryptophan auxotroph trp-28 is anomalous since preliminary mapping and suppression studies indicate the presence of a single amber nonsense mutation either late in trpE or early in trpD, but enzymological tests indicate the complete inactivation of both genes in this strain. Since the trpE and trpD genes are contiguous and encode the two subunits of a multifunctional enzyme complex, it was of interest to learn the mechanism of action of this apparent pleiotropic nonsense mutation. Our study has revealed that the phenotype of this strain derives not from a single mutation, but from the presence and interaction of multiple mutations. Besides the recognized amber mutation (designated trpD28), this strain carries two additional, conditionally expressed missense mutations (designated trpE1651 and trpD1652). The trpD28 amber codon maps in the promoter-proximal region 1 of trpD and eliminates the glutamine amidotransferase activity of the bifunctional trpD polypeptide. The trpD1652 mutation maps in the promoter-distal region 2 of trpD and severely reduces (but does not eliminate) the phosphoribosyl transferase activity of the trpD polypeptide. The trpE1651 mutation maps in the anterior part of trpE and causes a rapid loss of activity of the trpE polypeptide, but only when it exists as an uncomplexed subunit. The existence of the two missense mutations escaped prior notice in standard recombinational tests since the nature of each mutation is such that neither is detectable by the nutritional screens normally used in such tests unless an unsuppressed chainterminating mutation, such as trpD28, is also present.     

Complementation of a trpE deletion in Escherichia coli by Spirochaeta aurantia DNA encoding anthranilate synthetase component I activity.

A 2.7-kilobase Sau3A fragment of Spirochaeta aurantia DNA cloned in pBR322 complemented a trpE deletion in Escherichia coli. Deletion analysis and Tn5 mutagenesis of the resulting plasmid pBG100 defined a 2-kilobase-pair region that was required for both the complementation and the synthesis of 59,000- and 47,000-molecular-weight polypeptides (59K and 47K polypeptides) in maxicells. Both the 59K and the 47K polypeptides appear to be encoded by a single gene. A maxicell analysis of pBG100::Tn5 mutants suggests that the 47K polypeptide is not sufficient for the trpE complementation. In vitro and in vivo anthranilate synthetase (AS) assays indicate that the complementing activity encoded by pBG100 was functionally analogous to the AS component I of E. coli in that it utilized NH3 but not glutamine as the amino donor. pBG100 did not encode a glutamine amidotransferase activity, although the AS component I it encoded was capable of interacting with E. coli AS component II to catalyze the glutamine-requiring reaction. Expression appeared to depend on a promoter in the cloned S. aurantia DNA.     

Suppression of a deletion mutation in the glutamine amidotransferase region of the Salmonella typhimurium trpD gene by mutations in pheA and tyrA.

Prototrophic revertants of a trpD deletion mutant that lacks the glutamine amidotransferase domain of the bifunctional component II subunit of the anthranilate synthetase-phosphoribosyltransferase complex have been found to arise by the occurrence of sublethal missense mutations in either the pheA or tyrA loci. Such suppressor mutations were obtained directly by mutation of the wild-type pheA gene as well as indirectly by partial reversion of a variety of nonleaky pheA and tyrA mutations. The suppressor strains have only a portion of the normal level of the pheA or tyrA enzyme activity and thus experience a partial limitation in the synthesis of phenylalanine or tyrosine. This limitation leads to a relaxation of end-product regulation of the phenylalanine- or tyrosine-specific enzymes of the common aromatic pathway and to the overproduction of the branch point intermediate, chorismic acid, which is one of the substrates of the anthranilate synthetase reaction. It is proposed that the high intracellular level of chorismic acid acts to elevate the non-physiological NH3-dependent anthranilate synthetase activity of the component I subunit, thereby eliminating the need for the glutamine amidotransferase activity of the component II subunit. Consistent with this is the finding that phenylalanine and tyrosine are specific inhibitors of growth of the pheA and tyrA suppressor strains, respectively, causing a shutdown of the overproduction of chorismic acid by reestablishing normal end-product control of the common pathway.     

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.      

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.     

Translational Coupling during Expression of the Tryptophan Operon of ESCHERICHIA COLI

E. coli trpE polar mutations are 10 time more polar on trpD gene expression than on downstream (trpC, B, or A) gene expression. This effects was shown to be the result of "translational coupling," in which efficient translation of trpE-trpD intercistronic punctuation region consists of overlapping stop and start codons, and the trpE and trpD gene products form a functional complex in the cell. In light observations and characteristics, several models for the mechanism of translational coupling are considered.     

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.     

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.     

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.     

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.     

Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a Glu-tRNAGln kinase

Gln-tRNA(Gln) is synthesized from Glu-tRNA(Gln) in most microorganisms by a tRNA-dependent amidotransferase in a reaction requiring ATP and an amide donor such as glutamine. GatDE is a heterodimeric amidotransferase that is ubiquitous in Archaea. GatD resembles bacterial asparaginases and is expected to function in amide donor hydrolysis. We show here that Methanothermobacter thermautotrophicus GatD acts as a glutaminase but only in the presence of both Glu-tRNA(Gln) and the other subunit, GatE. The fact that only Glu-tRNA(Gln) but not tRNA(Gln) could activate the glutaminase activity of GatD suggests that glutamine hydrolysis is coupled tightly to transamidation. M. thermautotrophicus GatDE enzymes that were mutated in GatD at each of the four critical asparaginase-active site residues lost the ability to hydrolyze glutamine and were unable to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) when glutamine was the amide donor. However, ammonium chloride rescued the activities of these mutants, suggesting that the integrity of the ATPase and the transferase activities in the mutant GatDE enzymes was maintained. In addition, pyroglutamyl-tRNA(Gln) accumulated during the reaction catalyzed by the glutaminase-deficient mutants or by GatE alone. The pyroglutamyl-tRNA is most likely a cyclized by-product derived from gamma-phosphoryl-Glu-tRNA(Gln), the proposed high energy intermediate in Glu-tRNA(Gln) transamidation. That GatE alone could form the intermediate indicates that GatE is a Glu-tRNA(Gln) kinase. The activation of Glu-tRNA(Gln) via gamma-phosphorylation bears a similarity to the mechanism used by glutamine synthetase, which may point to an ancient link between glutamine synthesized for metabolism and translation.     

Analysis of cloned DNA from Leptospira biflexa serovar patoc which complements a deletion of the Escherichia coli trpE gene.

To analyze the cloned region of the chromosome of the spirochete Leptospira biflexa serovar patoc which complemented a defect in the trpE gene of Escherichia coli, we performed a series of experiments involving subcloning, transposon mutagenesis, and maxicells. By subcloning into pBR322 we were able to isolate the Leptospira genes on a 9.7-kilobase pair plasmid (pYC6). Transposon mutagenesis with Tn5 identified a 2.8-kilobase pair region of this plasmid as being necessary to complement a trpE deletion mutation in E. coli. Transformation of plasmid pYC6 into E. coli cells deleted for trpE and the proximal end of trpD showed that the Leptospira DNA complemented both defects. A maxicell analysis of various transposon-induced mutations of the plasmid revealed that three proteins (53.5, 23.6, and 22 kilodaltons) were encoded by the 2.8-kilobase pair region of the Leptospira genome. Two different promoters controlled the production of these three proteins.     

Glutamine phosphoribosylpyrophosphate amidotransferase-independent phosphoribosyl amine synthesis from ribose 5-phosphate and glutamine or asparagine

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic l-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed.     

Characterization of the glutamyl-tRNA(Gln)-to-glutaminyl-tRNA(Gln) amidotransferase reaction of Bacillus subtilis

In Bacillus subtilis, the formation of glutaminyl-tRNA is accomplished by first charging tRNA(Gln) with glutamate, which is then amidated. Glutamine was preferred over asparagine and ammonia as the amide donor in vitro. There is a functional analogy of this reaction to that catalyzed by glutamine synthetase. Homogeneous glutamine synthetase, from either B. subtilis or Escherichia coli, catalyzed the amidotransferase reaction but only about 3 to 5% as well as a partially purified preparation from B. subtilis. Several classes of glutamine synthetase mutants of B. subtilis, however, were unaltered in the amidotransferase reaction. In addition, there was no inhibition by inhibitors of either glutamine synthetase or other amidotransferases. A unique, rather labile activity seems to be required for this reaction.     

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.     

Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti

Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied. Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant. The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation. Histology of the nodules induced by trpE and aro mutants exhibited striking similarities. Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone. Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency. A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation. The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted. These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S. meliloti. The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.     

Plasmid vectors containing the tryptophan operon promoter suitable for efficient regulated expression of foreign genes

Derivatives of plasmid pBR322 that are suitable for high-level expression of foreign genes have been constructed. The vectors contain the Escherichia coli tryptophan promoter, the trpE gene, and about 15% of the trpD gene. To obtain expression, foreign genes are fused to the trpD gene fragment. After induction of the trp operon with 3 beta-indolylacrylic acid, trp gene products increase at least 50-fold, to account for 55% of the newly synthesised protein and 30% of total protein in the cell.