Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.     

Naturally occurring sites within the Shigella dysenteriae tryptophan operon severely limit tryptophan biosynthesis.

We investigated the structural, functional, and regulatory properties of the Shigella dysenteriae tryptophan (trp.) operon in transduction hybrids in which the cysB-trp-region of Escherichia coli is replaced by the corresponding region from S. dysenteriae. Tryptophan biosynthesis was largely blocked in the hybrids, although the order of the structural genes was identical with that of E. coli. Nutritional tests and enzyme assays revealed that the hybrids produced a defective anthranilate synthetase (ASase). Deletion mapping identified two distinct sites in trpE, each of which was partially responsible for the instability and low activity of ASase. We also discovered a pleiotropic site trpP (S) that maps outside the structural gene region and is closely linked to the S. dysenteriae trp operator. trpP (S) reduced the rate of trp messenger ribonucleic acid synthesis, and consequently trp enzyme levels, 10-fold relative to wild-type E. coli. In recombinants in which the structural genes of E coli were under the control of the S. dysenteriae promoter, enzyme levels were also reduced 10-fold. In some fast-growing revertants of the original hybrids, the rates of trp messenger ribonucleic acid synthesis and levels of tryptophan synthetase were restored to values characteristic of wild-type E.coli. Thus, the Trp auxotrophy associated with the S dysenteriae trp operon derives from the combination of a defective ASase and decreased expression of the entire operon imposed by trpP (S).     

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.     

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.     

Analysis in vivo of factors affecting the control of transcription initiation at promoters containing target sites for trp repressor

An investigation of repression in the trp system of Escherichia coli was undertaken using operon fusions and plasmids constructed via recombinant DNA technology. The promoters of the trp operon and the trpR gene were fused to lacZ, enabling the activity of these promoters to be evaluated under various conditions through measurements of beta-galactosidase production. In confirmation of earlier studies, the trpR gene was shown to be regulated autogenously. This control feature of the trp system was found to maintain intracellular Trp repressor protein at essentially invariant levels under most conditions studied. Increasing the trpR+ gene dosage did not significantly elevate Trp repressor protein levels, nor did the introduction of additional operator "sinks" result in significantly decreased levels of Trp repressor protein. Definite alterations in intracellular Trp repressor protein levels were achieved only by subverting the normal trpR regulatory elements. The placement of the lacUV5 or the lambda PL promoters upstream of the trpR gene resulted in significant increases in repression of the trp system. Substituting the primary trp promoter/operator for the native trpR promoter/operator resulted in an altered regulatory response of the trp system to tryptophan limitation or excess. The regulation of the trpR gene effectively imparts a broad range of expression to the trp operon in a manner finely attuned to fluctuations in intracellular tryptophan levels.     

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.     

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.     

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.     

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.     

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria

The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.