Letter to the editor: Characterization of mutations in the tryptophan operon of Escherichia coli by RNA nucleotide sequencing

The nucleotide sequences of operator-proximal tryptophan messenger RNA of Escherichia coli from strains with mutations very early in trpE have been determined. A frameshift mutant, trpE9777fs, has an additional A residue in the region coding for amino acids 4 and 5 of the trpE polypeptide. The other mutant, trpE9914am, exhibits an amber codon corresponding to the codon normally specifying amino acid residue 9 (glutamic acid) of the trpE polypeptide. These results demonstrate that the site of the trpE9777fs mutation precedes that of the trpE9914am mutation by 9 to 15 nucleotides.     

Milk Fat Globule Membrane Proteins in Aseptically Packed Ultra-heat-treated Milk: Changes during Storage

Five genes for tryptophan biosynthesis, trpE, trpD, trpC, trpB, and trpA of Brevibacterium lactofermentum, a coryne form glutamic acid-producing bacterium, were cloned as a 9.6 kb BamHl DNA fragment by colony hybridization. A previously cloned 1.2 kb Pst I DNA fragment containing a major part of the trpE gene was used as a probe. By complementation tests using the subclones of this 9.6 kb BamHl fragment and various tryptophan auxotrophs of B. lactofermentum and Escherichia coli, this fragment was found to contain a gene cluster composed of trpE, trpD, trpC, trpB, and trpA in this order. It suggests that genes for tryptophan biosynthesis in B. lactofermentum may be an operon.     

Localization of Two Functions of the Phosphoribosyl Anthranilate Transferase of Escherichia coli to Distinct Regions of the Polypeptide Chain

The trpD gene specifies a polypeptide which has both glutamine amidotransferase and phosphoribosyl anthranilate (PRA) transferase activities. Deletions fusing segments of trpD to the gene preceding it in the operon, trpE, were selected in strains carrying various trpD point mutations. The selection procedure required both that a deletion enter trpE and that it restore the PRA transferase activity which the parent trpD point mutant lacked. Deletion mutants were found which had PRA transferase activity although the first third of trpD was deleted. The existence of the mutants proves that a terminal segment of trpD is sufficient to specify a polypeptide having PRA transferase activity. The location of the deletion end points on the genetic map of trpD defines the extent of the trpD segment required for PRA transferase activity. This segment did not overlap the initial region of trpD required to specify the glutamine amidotransferase function of the trpD polypeptide. These results support the hypothesis (M. Grieshaber and R. Bauerle, 1972; H. Zalkin and L. H. Hwang, 1971) that the bifunctional trpD polypeptide might have evolved by fusion of a gene specifying a glutamine amidotransferase with a gene directing PRA transferase synthesis.     

Spontaneous, ultraviolet and ionizing radiation mutagenesis in two auxotrophic strains of Salmonella typhimurium carrying an R plasmid.

Ultraviolet-induced, gamma-induced and spontaneous mutation yields were studied in two different auxotrophic strains of Salmonella typhimurium in the presence and absence of the UV-protecting drug resistance transfer factor R-Utrecht. One strain, carrying the hisC527 (amber) mutation, showed significantly increased spontaneous, UV- and gamma-induced mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R factor, but appeared to show no significant increase in spontaneous mutability and only a very slight increase in gamma-mutability when carrying the R factor. These results demonstrate that the R-Utrecht plasmid, known to enhance UV-induced mutation yields in S. typhimurium, can also significantly enhance both spontaneous and gamma-induced mutation yields in this species. The latter effects are not so discernible with all markers, however, as shown by the results with strains carrying the trpD1 mutation. Enhancement of spontaneous mutability thus appears to be correlated with enhancement of gamma-mutability rather than UV mutability.     

Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.Translation termination is mediated by one of the three stop codons (UAA, UAG, or UGA). When such stop codons arise in coding sequences due to mutations, referred to as nonsense mutations, they lead to abrupt arrest of the translation process. However, the termination efficiency of such nonsense codons is not 100%, as certain tRNAs have the ability to read these nonsense codons. Genetic code ambiguity is seen in several organisms. Stop codons have been shown to have alternate roles apart from translation termination. In organisms from all three domains of life, UGA encodes selenocysteine through a specialized mechanism. In Methanosarcinaceae, UAG encodes pyrrolysine (3). UAA and UAG are read as glutamine codons in some green algae and ciliates such as Tetrahymena and Diplomonads (24), and UAG alone encodes glutamine in Moloney murine leukemia virus (32). UGA encodes cysteine in Euplotes; tryptophan in some ciliates, Mycoplasma species, Spiroplasma citri, Bacillus, and tobacco rattle virus; and an unidentified amino acid in Pseudomicrothorax dubius and Nyctotherus ovalis (30). In certain cases the context of the stop codon in translational readthrough has been shown to play a role; for example, it has been reported that in vitro in tobacco mosaic virus, UAG and UAA are misread by tRNA^Tyr in a highly context-dependent manner (34, 9).Termination suppressors are of three types, i.e., amber, ochre, and opal suppressors, which are named based on their ability to suppress the three stop codons. Amber suppressors can suppress only amber codons, whereas ochre suppressors can suppress ochre codons (by normal base pairing) as well as amber codons (by wobbling) and opal suppressors can read opal and UGG tryptophan codon in certain cases. As described by Sambrook et al. (27), a few amber suppressors can also suppress ochre mutations by wobbling. The suppression efficiency varies among these suppressors, with amber suppressors generally showing increased efficiency over ochre and opal suppressors. supE44, an amber suppressor tRNA, is an allele of and is found in many commonly used strains of Escherichia coli K-12. Earlier studies have shown that supE44 is a weak amber suppressor and that its efficiency varies up to 35-fold depending on the reading context of the stop codon (8).Translational accuracy depends on several factors, which include charging of tRNAs with specific amino acids, mRNA decoding, and the presence of antibiotics such as streptomycin and mutations in ribosomal proteins which modulate the fidelity of the translational machinery. Among these, mRNA decoding errors have been reported to occur at a frequency ranging from about 10⁻³ to 10⁻⁴ per codon. Translational misreading errors also largely depend on the competition between cognate and near-cognate tRNA species. Poor availability of cognate tRNAs increases misreading (18).Several studies with E. coli and Saccharomyces cerevisiae have shown the readthrough of nonsense codons in suppressor-free cells. In a suppressor-free E. coli strain, it has been shown in vitro that glutamine is incorporated at the nonsense codons UAG and UAA (26). It has been reported that overexpression of wild-type tRNA^Gln in yeast suppresses amber as well as ochre mutations (25). In this study, we have confirmed the presence of an amber suppressor mutation in the glnX gene in a supE44 strain by sequence analysis. This was done essentially because we observed that supE44 could also suppress lacZ ochre mutations, albeit inefficiently. On further investigation using an in vivo luciferase reporter assay system for tRNA-mediated nonsense suppression (28), we found that the efficiency of suppression of amber lesion by supE44 is significantly higher than that reported previously in the literature. An increased ability to suppress ochre and opal nonsense mutations was observed in cells bearing supE44 compared to in the wild type. Such an effect was observed only in the stationary phase and was abolished in logarithmic phase.     

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.     

Identification of novel alleles induced by EMS-mutagenesis in key genes of kernel hardness and starch biosynthesis in wheat by TILLING

To identify novel allelic variations in key genes of wheat quality, the present study used the targeting induced local lesions in genomes platform to detect point mutations in target genes. The wheat variety Longfumai 17 was treated by the mutagen ethyl methanesulfonate to produce a bulk M₂ generation, and the population included 1122 plants. A total length of 3906.80 kb nucleotides was analyzed, and the average mutation density was 1/244.17 kb. The identified mutations included G>A substitutions (43.75%), C>T substitutions (31.25%), A insertions (12.50%), T insertions (6.25%), and deletions (6.25%). These point mutations led to changes in amino acids and thus the encoded protein sequences, ultimately producing 18.75% of missense mutations, 12.50% of frame shift mutations, 6.25% of nonsense mutations, 25.00% of silent mutations and 37.50% of non-coding region mutations. In the kernel hardness gene Pinb and 3 starch synthesis genes waxy, Agp2 and SSIIa-A, we detected 16 different point mutations in 25 mutant lines. The Pinb gene harbored two missense mutations and a nonsense mutation; the C>T missense mutation resulted in a novel allele, this novel allele and the nonsense mutation alerted protein 3D structure; the waxy gene presented missense and frame shift mutations; the Agp2 gene carried a missense mutation; the SSIIa-A incurred a missense mutation and a frame shift mutation that resulted in premature protein termination. All the frame shift mutations, nonsense mutations and the Pinb novel allele resulted in allelic variation of their corresponding genes, which in turn affected their gene functions. The identified mutant lines can be used as intermediate materials in wheat quality improvement schemes.     

Polarity suppressors increase expression of the wild-type tryptophan operon of Escherichia coli

Three new polarity suppressors, selected to relieve the polar effect of nonsense mutations in the tryptophan (trp) and lactose (lac) operons of Escherichia coli, increase expression distal to nonsense mutations in both operons to a greater extent than suA. These suppressors relieve the polarity created by amber, ochre and frameshift mutations with equal efficiency.Two of the three polarity suppressors elevate enzyme synthesis in the wildtype trp operon two- and fivefold, respectively. The increase in enzyme levels is in each case correlated with increased levels and rates of synthesis of structural gene trp messenger RNA. Since expression of all genes is elevated, these findings suggest the existence of a site early in the wild-type trp operon that affects the extent of operon expression. We located the site affected by these two polarity suppressors between the operator and the first structural gene, trpE. Although the third polarity suppressor also relieves mutational polarity efficiently, it has no detectable effect on expression of the wild-type trp operon.     

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.     

Untranslated T7 Phage mRNA is stabilized in <Emphasis Type="Italic">suA</Emphasis> Host

IT has been shown¹ that relief from the polar effect of nonsense mutations in the tryptophan Operon by the suA allele² is associated with increased survival of mRNA operator distal to the nonsense mutation. Experiments described here confirm the effect of suA on mRNA distal to amber mutations and directly demonstrate the complete mRNA molecule only in the presence of the suA mutation.     

Mutators in SACCHAROMYCES CEREVISIAE: MUT1–1, MUT1–2 and MUT2–1

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.     

The reversion of trp frameshift mutations in mut,polA, lig and dnaE mutant strains of Escherichia coli

Mutator mutations mutL25, mutR34, and mutU4 had similar effects on the reversion of 4 trp frameshift mutations of known sequence. The mutation trpE9777, which resulted from the addition of an A–T base-pair to a run of 5 A–T base-pairs, was most strongly reverted by the 4 mutators. Reversion of trpE9777 was also increased by mutation polA1 (DNA polymerase I) and dnaE486 and dnaE511 (DNA polymerase III). No effect was found with the ligase mutations, lig-4 or lig-ts7. Mutations polAex1 and polA107, both deficient in the 5′ → 3′ exonuclease activity of DNA polymerase I, had different mutator effects; the factor increase in reversion of trpE9777 was 28-fold for polAex1, 6-fold for polA107, and 21-fold for polA1. The trpE9777 mutation is a useful indicator of frameshift mutator activity.     

Genetic and Physiological Characterization of met15 Mutants of SACCHAROMYCES CEREVISIAE: a Selective System for Forward and Reverse Mutations

One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus.—It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.     

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.     

Gene structure in the tryptophan operon of Escherichia coli: Nucleotide sequence of trpC and the flanking intercistronic regions

We have determined the DNA sequence for the portion of the Escherichia coli tryptophan (trp) operon spanning trpC, which codes for the bifunctional enzyme N-(5′-phosphoribosyl)-anthranilic acid isomerase/indole-3-glycerol phosphate synthetase. The coding region consists of 1356 nucleotides, directing the synthesis of a polypeptide 452 amino acids in length. The predicted protein sequence is consistent with the amino acid composition of the pure enzyme, and with all known partial peptide sequences derived from this molecule. The enzyme is of particular functional interest, because it contains the catalytic activities for two sequential reactions in tryptophan biosynthesis in a single polypeptide chain.The nucleotide sequences of the junctions between trpC and its flanking genes, trpD and trpB, have also been determined. The trpD-trpC junction consists of six untranslated nucleotides and translation of trpC initiates at the second of two adjacent AUG codons. The trpC termination codon is separated from trpB by 11 nucleotides. The short non-translated regions flanking trpC distinguish it from trpA and trpD, whose initiation codons overlap the termination codons of the preceding genes (trpB and trpE), respectively. These differences in the intercistronic regions may reflect functional relationships between the products of adjacent genes in the operon.     

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.     

Nucleotide sequences of the trpG regions of Escherichia coli,Shigella dysenteriae,Salmonella typhimurium and Serratia marcescens

The nucleotide sequences of Serratia marcescens trpG and the corresponding regions of Escherichia coli, Shigella dysenteriae and Salmonella typhimurium trpD have been determined. Analysis of the nucleotide sequence divergence suggests the following evolutionary relationships: Serratia-[Salmonella, (Escherichia, Shigella)]. Partial reconstruction of ancestral nucleotide sequences and subsequent analysis of nucleotide substitutions show that the majority of nucleotide substitutions in the evolution of trp(G)D are transitions that result in a reduction of G + C content. Since most of the nucleotide substitutions are in the third position of codons, bias in synonymous codon usage also reflects G + C content. The trpE-trp(G)D junction in the four organisms is characterized by overlapping translation termination and initiation codons. The relative positions of trpE and trp(G)D thus became fixed in evolution before the fusion of trpG and trpD. Nucleotide sequences representing the fusion of trpG and trpD in Escherichia, Shigella and Salmonella are not more nor less divergent than other portions of the trp(G)D coding sequences.