Tryptophan genes in Rhizobium--their organization and their transfer to other bacterial genera.

Summary R. leguminosarum trp alleles mapped by R68.45-mediated recombination were located in three distinct chromosomal regions. We isolated three derivatives of R68.45 that carried different trp genes of R. meliloti. Each of the plasmids suppressed all of the R. leguminosarum trp alleles in a particular region. The R-primes were transferred to strains of P. aeruginosa carrying mutations in different trp genes. The plasmid pAJ24JI suppressed trpA, B and F mutants, pAJ73JI suppressed trpC and D and pAJ88JI suppressed a trpE mutant. When the R-primes were transferred to E. coli trp strains they failed to suppress any trp mutants. A derivative of pAJ24JI was isolated which was able to suppress trpA and F mutants of E. coli.     

Re-initiation of tryptophan operon expression in a promoter deletion strain of Salmonella typhimurium.

Summary A genetic and enzymological study was made of five spontaneous prototrophic revertants of a tryptophan auxotroph of Salmonella typhimurium which carries a deletion extending from the closely linked supX locus into the trp operator-promoter region. The revertants were found to have regained initiation of expression of all five trp genes. Recombinational tests showed that in each case the genetic change responsible for re-initiation is cotransducible with the trp-cysB region of the chromosome. Two different mechanisms leading to re-initiation of trp gene expression were established: (a) an extension of the limits of the original deletion resulting in the fusion of the trp structural genes with a nearby gene or gene set located outside the operator end of trp, and (b) translocation of a duplicate set of the trp structural genes to other chromosomal sites, located operator-distal to the normal trp operon, in such a manner that they are functionally fused to foreign genetic units. One revertant which arose by mechanism (a) was shown to have an extended deletion with one new terminus in trp and the other in the nearby cysB locus. All the revertants exhibit constitutive expression of the trp enzymes, with activities varying among strains from five to forty five times greater than the fully repressed wild type level. The protein product of trpA, the first structural gene of the operon, appears to have been partially damaged by the re-initiation event in at least two strains, while in the other strains, the enzyme appears in preliminary tests to be indistinguishable from that of wild type.     

Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

Summary RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp^– phenotype to Trp⁺. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.     

Genetic fusions that help define a transcription termination region in Escherichia coli

The trpA gene product was analyzed from a class of strains of Escherichia coli K12 in which the lac operon has been fused by deletion to the trp operon. These are strains that have retained the ability to synthesize tryptophan. Two of these strains are shown to make a wild-type trpA product; these strains retain intact all structural genes of the ttrp operon. It is proposed that the lac operon in these strains is fused to a region of the trp operon between trpA, the last gene in the operon, and the region where trp messenger RNA synthesis terminates. The region where trp messenger RNA synthesis terminates thus is distinct from the trp structural genes.     

The influence of amino acid starvation on the UV reversibility of the strains ofEscherichia coli B/rthy − trp − Hcr + andEscherichia coli B/rthy − trp − Hcr −

The fraction of inducedtrp ⁺ reversions in the strains ofEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁺ andEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁻ was studied in the course of starvation for an essential amino acid. UV light as a mutagenic factor was used. It was found that there is a decrease in the proportion of inducedtrp ⁺ reversions in the strain ofHcr ⁺ type during starvation. Such a decrease was however observed only with that fraction oftrp ⁺ reversions which is expressed in selective plates where several divisions of irradiated cells are caused. The proportion oftrp ⁺ reversions expressed on minimal plates does not change during starvation. With the strain ofHcr ⁻ type the proportion of inducedtrp ⁺ mutations remains unaltered irrespective of the nature of the selective plates.     

Expression of the tryptophan operon in merodiploids of Escherichia coli. I. Gene dosage, gene position and marker effects

Summary Under conditions of derepression,Escherichia coli K12 strains diploid for thetrp operon specify more than twice as much enzyme as a haploid. The disproportionate increase probably occurs because episomally carriedtrp genes tend to specify more enzyme than do chromosomal genes.Operons harboring the nonsense mutationtrpA2 or the missense mutationtrpBYS-101 specify less protein than do wild-type operons. This effect varies with operon location in the case oftrpBYS-101.In a homozygoustrp merodiploid A46/F A46 reversion totrp ⁺ occurs three times as frequently in episomal DNA as in chromosomal DNA. Thus, if the chromosome: Ftrp episome ratio inE. coli is one, as demonstrated by Helinski and co-workers, the rate of gene expression and the rate of mutation can vary and depends upon the location of the DNA within the cell.Supported by Grant AM-12150 from the National Institutes of Health. Journal Paper No. 3973 of Purdue Agricultural Experiment Station.     

Decay rates of Escherichia coli trp messenger RNA molecules lacking the normal 5''-terminal sequences.

Summary We have examined the stability in vivo of three mutant species of trp mRNA which differ from wild type in the nature of their 5-termini. These novel mRNA molecules originate from three mutationally generated promoters which lie within trp structural genes: trpE1423 lies near the carboxy-terminal end of trpE, trpD11 lies near the carboxy-terminal end of trpD (McPartland and Somerville, 1976) and trpC2121 lies near the center of trpC. When mRNA synthesis from the wild-type promoter is repressed by tryptophan, these strains still synthesize trp mRNA from their internal promoters at a relatively high efficiency in a constitutive fashion. The trp mRNA thus produced by the mutants lacks various lengths of the wild-type 5-proximal RNA sequence. These shortened trp mRNA molecules decayed exponentially, at about the same rate as that of normal trp mRNA whose synthesis originates at the authentic trp promoter. Chloramphenicol inhibited the degradation of 5-truncated trp mRNA fragments in a manner similar to that observed for wildtype trp mRNA, suggesting that the usual mechanism of mRNA decay is operative. We postulate that the initiation of mRNA decay at or near the 5-end does not require some special nucleotide sequence; rather the 5-proximal protion in general constitutes the target for nucleolytic attack.     

Co-operative binding of two Trp repressor dimers to α- or β-centred trp operators

The α-centred trp operator binds one dimer of the Trp repressor, whereas the β-centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr-Gly-7 substitution binds almost as well as the wild-type Trp repressor to the α-centred trp operator, but it does not bind to the β-centred trp operator. This confirms that Tyr-7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with a-centred trp operator variants showed that positions 1 of the a-centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the α-centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T-A to G-T) effectively transform the α-centred trp operator into a β-centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a β-centred trp operator.     

Modulation of Escherichia coli tryptophan (trp) attenuation by the UGA readthrough process

Summary We constructed plasmid pAtrp46 in which lacZ gene expression is regulated by the attenuator of the Escherichia coli tryptophan (trp) operon. The attenuation of trp, which occurs in the presence of an excess of tryptophan, is reflected by a decrease in the expression of the lacZ gene of pAtrp46 in a trpR^- strain. Experiments with pAtrp46 further support our previous results (Engelberg-Kulka et al. 1982b) that suppression of a UGA termination codon by normal charged tRNA^Trp, a process called UGA readthrough, is a necessary mechanism in trp attenuation. Our experiments also suggest that plasmid pAtrp46 is useful for studies of other aspects of trp attenuation.     

Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis

Summary Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the -galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.     

The use of specialised transducing phages in the amplification of enzyme production

Two types of trp phages have been used as model systems to investigate ways of optimising the expression of bacterial genes from transducing phage genomes.Excellent yields of trp enzymes were achieved by infecting a trpR^– host with Q ^– or Q ^– Q ^– S ^– derivatives of trpAM1, which expresses its trp genese exclusively from the trp promoter. The five trp geneproducts constituted more than 50% of the total soluble protein of infected cells under these conditions, and an even higher proportion of the protein synthesized after infection. In a trpR ⁺ host, phage DNA replication was easily able to override tryptophan-mediated repression by titration of the trp repressor protein. N ^– derivatives of trp phages carrying the trp promoter were equally productive, while having the advantage of being much simpler to construct and propagate.     

Amplification of the tryptophan operon gene in Escherichia coli chromosome to increase l-tryptophan biosynthesis

Classical mutagenesis could desensitize the feedback inhibition of l-tryptophan (l-Trp) biosynthesis. Among the mutants, a5-fluorotryptophan-resistant strain, Escherichia coli EMS4-C25 produced 3 g/l of l-Trp within 18 h. The feedback-resistant l-Trp operon gene (trp) prepared from E. coli EMS4-C25 was inserted into pUC19 and pHSG576 to generate pTC701 and pTC576, respectively. When pHSG576 and pTC701 were introduced into E. coli EMS4-C25, chromosomal integration occured through homologous recombination. By using Souther hybridization, we demostrated that the integrated plasmids existed as multicopies. The strains with integrated foreign trp operon gene had higher activities of anthranilate synthase and Trp synthase than those found for the host strain and produced 9.2 g/l of l-Trp with 13% conversion yield from d-glucose. The integration and implification of the trp-operon-beraing plasmid avoided the plasmid instability and increased l-TRp production. Correspondence to: E.-C. Chan     

Formation of F''TRP Plasmids in ESCHERICHIA COLI K12

From strains carrying two different F-prime factors, we recovered F' derivatives that acquired the trp chromosomal region. These F'trp plasmids can be isolated at a frequency of 10^-5 to 10^-6. They were characterized genetically by looking at the size of the trp segment they acquired and at the location of that segment in the parental F' plasmid. Results are discussed in relationship to possible transposition mechanisms.     

Elimination of lethal and pre-mutational DNA lesions during the photoreactivation of UV-irradiatedEscherichia coli

The comparison of the frequency oftrp ⁺ revertants ofEscherichia coli B/r Hcr⁺ thy trp after UV-irradiation on the one hand and after UV-irradiation plus photoreactivation on the other showed that both photoreversible pyrimidine dimers of the cyclobutane type and the non-photoreversible DNA lesions cause, at equal lethal effects, alsotrp ⁺ reversions with the same efficiency. If lethal, the pyrimidine dimers may thus be conceived as primary pre-mutational lesions.     

Direct identification of the amino acid changes in two mutant lac repressors.

Deletions extending into the trp operon at one terminus and the lacI control region at the other terminus have been examined. One of these, B116, ends within the trp leader sequence and eliminates the trp attenuator site, placing the synthesis of lac repressor under trp control. We have isolated and characterized the B116 repressor. The protein sequence of the aminoterminus of B116 shows that an additional 16 residues are added to the amino-terminal end of wild-type repressor. Moreover, a valine residue appears in place of methionine at position 17 (the original amino-terminal residue of the wild-type repressor). A comparison of the messenger RNA sequence of the trp leader region and of the I leader region demonstrates that the translation of the B116 repressor is initiated at an AUG codon within the trp leader sequence. The GUG initiation codon at the start point for translation of wild-type repressor is now read as valine, since it appears at an internal position (residue 17 of the altered repressor). The B116 repressor accumulates at levels as high as 1% of the soluble cell protein in trpR^? strains. The efficiency of the trp leader initiation codon in translation suggests that in wild-type strains this AUG is also active in directing protein synthesis, which would result in a polypeptide consisting of 14 amino acids. We have examined the physical properties of the B116 repressor, which shows a marked tendency to form higher aggregates. Other characteristics of B116 are also described.     

High expression vectors for the production of recombinant single-chain urinary plasminogen activator from Escherichia coli

Summary An expression cassette containing a synonymous gene for human single-chain urokinase-type plasminogen activator (Rscu-PA) 5'-flanked by a trp promoter and the Shine-Dalgarno sequence of the xyl A operon of Bacillus subtilis and terminated by the terminators trp A and Tn10 was constructed and inserted into a pBR322 derivative to yield pBF160. When compared to pUK54 trp 207-1 containing the natural scu-PA gene without the Shine-Dalgarno sequence and terminator, the expression efficiency of pBF160 in Escherichia coli strains was improved by one order of magnitude. Replacement of the trp by the tac promoter (pBF171) did not affect expression. Inserting the Shine-Dalgarno sequence and Tn10 terminator into pUK54 trp 207-1 (pWH1320) slightly increased the expression level, whereas elimination of the Shine-Dalgarno sequence and the terminators from pBF160 with almost complete conservation of the synonymous structural gene (pBF191) significantly reduced the expression. Variation of the distance between the Shine-Dalgarno sequence and the start codon between 8 and 10 bp (pBF163) proved irrelevant. In conclusion, poor expression of mammalian genes in E. coli may result from both improperly designed regulatory elements and structural features of the coding region and therefore de-novo synthesis of the gene may be required to obtain satisfactory expression.     

Thr region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function

Among a collection of 34 independent mutants with internal deletions in the trp operon of Escherichia coli we found six that fail to recombine with any known point mutant in trpE, the first gene in the operon. These six deletion mutants are regulated normally by tryptophan and thus appear to have the trp operator region intact. However, four of these deletions result in alterations in the maximum level of expression of the trpC, B and A genes when compared with wild type or with an internal deletion of similar length which retains a small operatorproximal segment of trpE. Two of these deletion mutants, trpΔED1 and trpΔED12, have lower levels of the protein products of trpB and trpA than the control strains. In contrast, deletions trpΔED2 and trpΔED102 both markedly increase the levels of the trpB and trpA polypeptides. Deletion mutant trpΔED2 has 3 to 3.5 times and mutant trpΔED102 has seven to eight times as much tryptophan synthetase β₂ and α proteins as the wild-type or deletion control strains. The increase in tryptophan synthetase β₂ and α proteins seen is a consequence of an increase in the level of trp mRNA directing the synthesis of these enzymes. The rate of synthesis of trpBA mRNA is increased in trpΔAED2 about twofold, and in trpΔED102 about four- to sixfold over the control strain. The left-hand deletion end-points of both trpΔED2 and trpΔAED102 have been shown to map to the right of a known trp operator-constitutive mutation and appear to lie before the first translation start codon in trpE (M. Bronson, C. Squires &; C. Yanofsky, unpublished results). We propose that these deletions alter a region between the earliest known trpE point mutation and the trp operator which influences the maximum rate of synthesis of trp operon mRNA.     

Increased spontaneous mutation rates in mutants of E. coli with altered DNA polymerase III

Summary Two temperature-sensitive mutants in dnaE, the structural gene for DNA polymerase III of Escherichia coli, show increased spontaneous mutation rates at permissive temperatures. Studies of the reversion of well-characterized trpA mutations in dnaE strains show that the mutagenic effect of altered DNA polymerase III applies to several different base substitution events, but not to frameshifts. The results suggest that DNA polymerase III is involved in base-selection during DNA replication.MRC Molecular Genetics Unit