A mutation to 5-methyltryptophan dependence in the trp operon of Salmonella typhimurium. III. Correlation between phenotype and the properties of the second enzyme for tryptophan biosynthesis in a 5-methyltryptophan dependent mutant and several 5-methyltryptophan-independent revertants.

Summary The AS-PRT enzyme complex which catalyzes the first two steps in the biosynthesis of tryptophan in S. typhimurium consists of two polypetide subunits: anthranilate synthetase (component I or AS-CoI) and anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT). These polypeptides are the products of the first two structural genes of the trp operon, trpA and trpB respectively. The PRT component has two functions: the aminoterminal 40% of the polypeptide is necessary for glutamine amidotransferase activity (GAT) while the carboxy-terminal 60% carries out the PRT activity proper. The mutant strain SO495 has a mutation, trpA515, which confers a unique phenotype: while the strain is capable of utilizing anthranilic acid (AA) a substrate of PRT, as a growth factor, it can only do so in the presence of the analogue 5-methyltryptophan (MT) normally a potent growth inhibitor. Previous evidence indicates that SO495 may possess a somewhat altered PRT, and that its activity could be inhibited by an altered, enzymatically inactive AS made in this strain under derepression. Some experiments designed to test these possibilities are described in this paper. Various properties of the PRT's of the MT-dependent mutant and several of its MT-independent revertants were examined and compared. These included the determination of their apparent Km's for the substrates anthranilic acid (AA) and phosphoribosyl pyrophosphate (PRPP) and the presence or absence of GAT activity. In addition, the possibility that a complex consisting of PRT and an enzymatically inactive AS-CoI was present in some of the revertant strains only when grown under derepressing conditions was investigated by gel chromatography. The results showed that the MT-dependent strain SO495 and the MT-independent revertants have PRT's which differ from each other as well as from wild type LT7 PRT. In MT-independent revertants which retain the trpPO region and most of trpA, PRT can form a loose aggregate which elutes from Bio-Gel columns as three fast moving peaks. This loose aggregate is absent when the strains are grown under repressing conditions and is always absent in strains which lack most of the trpA gene. These results support the idea that the dependence of strain S0495 on MT for utilization of AA as a growth factor has to do with the inhibition of the altered PRT made in this mutant by an altered AS-CoI polypeptide which is synthesized only under derepression. They also suggest that translation of trpB starts from different points in the wild type, S0495 and the MT-independent revertants.     

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.     

Deletion induction in bacteria. I. The role of mutagens and cellular error-prone repair

The amber mutation trpD28 of Salmonella typhimurium shows a complex reversion pattern on anthranilate (AA)-supplemented minimal medium. Under such conditions it is possible to recover revertants of two phenotypes, prototrophs (MM+) and anthranilate utilizers (AA+), each phenotype brought about by several mutational events. Since one class of AA+ revertants is caused by deletion of the trpD28 mutation, this constitutes a useful system for quantitative studies of the effects of mutagenic agents and cellular factors on the production of deletions. In the present study we have tried to assess the relative contribution of chemical mutagens vs. cellular mutator factors in causing this class of mutations. Strains of S. typhimurium in which the spontaneous reversion rate of trpD28 was modified by pKM101, (strain SO1007), mutL (strain SO1018) and both (strain SO1008), as well as the wild type (strain SO939) were treated with nitrous acid (HNO2) and mitomycin C (MC), mutagens reported to induce deletions in bacteria. The results showed that while the absolute frequency of deletions increased exponentially with dose of mutagen in parallel with the total reversion frequency, the relative frequency (percent) of these mutations was characteristic for each strain and for the most part unaffected by the dose of mutagen. It appears that deletions, spontaneous or induced, occur as a fixed percentage of total mutations and are brought about by the cells' own repair capacity and characteristic DNA metabolism. Perhaps these mutations are the result of untargeted events during SOS misrepair.     

“Self-Feeding” Strain of Salmonella typhimurium with a Mutation in the trpB Gene and Nutritional Requirements of trpA Gene Mutants

An indole-requiring (Ind(-)) mutant of Salmonella typhimurium, isolated from a culture of a leaky trpA mutant, was genetically analyzed by P22-mediated transduction. The mutation site giving the Ind(-) phenotype was shown to be in trpB, the second gene of the trp operon. A second mutation at this site resulted in change of nutritional requirement from indole to anthranilic acid (Anth(-)). This phenotype is normally associated with mutations in the first trp gene, trpA. However, the Anth(-) mutant also excreted anthranilic acid and showed "self-feeding" on unsupplemented media. Of two possible explanations for this aberrant phenotype, the first, that the trpB mutations may be in the "unusual" region, was dismissed on genetic evidence and on the biochemical evidence that an active anthranilate synthetase (AS) is produced. The alternative explanation, that the affected enzymatic activity, phosphoribosyl transferase, is unstable in vivo, but its AS component 2 activity is stable, is considered more probable.     

Reversion of trpA nonsense mutations by deletion of the chain-termination codons

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.     

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.     

A new strain of Salmonella typhimurium reverted by mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine--a possible universal tester for mutagenic compounds

A strain of Salmonella typhimurium, SO1007, which carries the amber mutation trpD28 plus the plasmid pKM101 was reverted very efficiently by two mutagens with different mutagenic specificities and modes of action: mitomycin C (MC) and N-methyl-N'-nitro-N-nitrosoguanidine (NG). By selecting revertants on minimal agar supplemented with anthranilic acid (AA), two distinct phenotypic classes of TrpD28 revertants can be recovered: prototrophs (MM+) and anthranilate utilizers (AA+). Since each phenotypic class is known to be caused by a variety of mutational events, reversion of trpD28 on minimal-anthranilate medium may be useful for detecting mutagenic agents regardless of the types of mutations they may cause. Thus, strains like SO1007 may be useful as 'universal' detectors of mutagenic compounds. In the course of these experiments we also observed that pKM101 does not protect but, on the contrary, sensitizes the host bacteria slightly to the toxic effects of MC.     

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.     

Transfer of episome F'lac+ and chromosomal trp+ genes from Erwinia amylovora to Salmonella typhimurium.

The F'lac+ episome of Escherichia coli origin was transferred by conjugation with frequencies of 10(-7) to 10(-5) from Erwinia amylovora to 14 out of 15 Salmonella typhimurium trp female parents. The chromosomal trp+ genes were transferred with frequencies of 10(-7) to 10(-6) only to one trpB and 2 trpD female parents, which have a point mutation in the 2nd and fourth structural genes, respectively, of the tryptophan operon. The transferred male trp+ genes became integrated at the selected sites of the S. tryphimurium chromosome. The resulting Trp+ hybrids were phenotypically stable, lacked a cryptic trp allele selected against in the female parent, had high genetic homology values in the tryptophan region, and showed biochemical reactions and pathogenicity typical of S. typhimurium.     

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.     

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.     

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.     

Antimutagenic effects of chemicals on mutagenesis resulting from excision of a transposon in Salmonella typhimurium

We have found that a temperature-sensitive mutation in the polA gene of Salmonella typhimurium strain LT2 causes precise excision of transposon Tn10 to occur at significantly increased frequencies in cells incubated at the restrictive temperature. In our experiments, precise excision from a site in the tryptophan operon was measured by determining the frequency of reversion of the auxotrophic trp1014::Tn10 polA7 strain to prototrophy on defined medium containing a trace amount of broth. Because the yields of revertants at 37 degrees C were of the order of 200 colonies per plate, it was possible to measure the effects of chemical inhibitors on the processes involved in precise excision. We now report that all of the DNA-repair inhibitors we have studied (caffeine, ethionine, acriflavine, procaine and cinnamaldehyde) are effective inhibitors of precise excision of Tn10, and can therefore be defined as antimutagens.     

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.     

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.