Genetic and physical analyses of Caulobacter crescentus trp genes.

Caulobacter crescentus trp mutants were identified from a collection of auxotrophs. Precursor feeding experiments, accumulation studies, and complementation experiments resulted in the identification of six genes corresponding to trpA, trpB, trpC, trpD, trpE, and trpF. Genetic mapping experiments demonstrated that the trp genes were in two clusters, trpCDE and trpFBA, and a 5.4-kilobase restriction fragment from the C. crescentus chromosome was isolated that contained the trpFBA gene cluster. Complementation experiments with clones containing the 5.4-kilobase fragment indicated that trpF was expressed in Escherichia coli and that all three genes were expressed in Pseudomonas putida. This expression was lost in both organisms when the pBR322 tet gene promoter was inactivated, indicating that all three genes were transcribed in the same orientation from the tet promoter. Thus, the C. crescentus promoters do not seem to be expressed in E. coli or P. putida. Complementation of the C. crescentus trp mutants indicated that the tet promoter was not necessary for expression in C. crescentus and suggested that at least two native promoters were present for expression of the trpF, trpB, and trpA genes. Taken together, these results indicate that C. crescentus promoters may have structures that are significantly different from the promoters of other gram-negative species.     

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.     

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.     

Tryptophan of facultative methylotrophic bacteria Pseudomonas sp. M. II. Regulation of tryptophan biosynthesis

Regulation of tryptophan biosynthesis of facultative methylotrophic Pseudomonas sp. M was studied. Repression of the trpE, trpD and trpC genes by tryptophan was demonstrated. It was also shown that the trpE and trpDC genes are derepressed noncoordinately. No regulation of the trpF gene product could be demonstrated, indicating that its synthesis is constitutive. The trpA and trpB genes are inducible by indol-3-glycerophosphate. Anthranilate synthase and tryptophan synthase were sensitive to the feedback inhibition. The tryptophan concentrations giving 50% inhibition were estimated to be 9 microM and 1 microM, respectively. Experimental evidence for activation of the N-5-phosphoribosyl anthranilate isomerase and for inhibition of the indol-3-glycerophosphate synthase by some tryptophan intermediates was obtained.     

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.     

The tryptophan operon of the facultative methylotrophic bacteria Pseudomonas sp. M. III. Characteristics of regulatory 5-MTR mutants

Regulatory 5-DL-methyltryptophan (5-MT)-resistant mutants of facultative methylotrophic Pseudomonas sp. M. were obtained. They are able to excrete tryptophan into the growth medium (60 to 300 g/ml). 5-MTR regulatory mutants are characterized by depression of trpE, trpD and trpC genes, which causes the production of intermediates of tryptophan biosynthesis and results in trpA and trpB genes induction as well as in two-fold activation of N-5-phosphoribosyl anthranilateisomerase (trpF gene product). Besides, all mutants demonstrate reduction of synthase feed-back inhibition about 4-11-fold. Together with tryptophan excretion, 5-MTR regulatory mutants are able to excrete tyrosine and unable to utilize this amino acid as the sole carbon source, which points to multiple nature of the selective effect of 5-MT.     

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.     

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.     

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.     

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.     

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.     

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.      

Mitomycin C-induced expression of trpA of Salmonella typhimurium inserted into the plasmid ColE1.

EcoRI endonuclease digestion of the deoxyribonucleic acid of a phi80 transducing phage carrying the entire tryptophan (trp) operon of Salmonella typhimurium (phi80 S.t.trpE-A) yielded a 4.3 X 10(6)-dalton fragment containing intact trpE, trpD, and trpC and a 3.35 X 10(6)-dalton fragment containing intact trpA. The trpA fragment inserted into EcoRI-cleaved plasmids ColE1 and CR1 was expressed regardless of its orientation of insertion. Mitomycin C, a compound that induces colicin E1 production in ColE1-containing bacteria, stimulated tryptophan synthetase alpha production in cells containing ColE1-TRPA plasmids with the trpA fragment inserted in one orientation but not the other. We conclude that in the inducible plasmids trpA can be expressed from the colicin E1 promoter.     

Regulation of tryptophan genes in Rhizobium leguminosarum.

Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically. They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan. Missing enzyme activities allowed assignment of all mutant to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol. Gen. Genet. 165:323-330, 1978). In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.     

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.     

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.     

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.