Regulatory mutants of the aroF-tyrA operon of Escherichia coli K-12.

The regulatory region of the aroF-tyrA operon was fused to the chloramphenicol acetyltransferase (cat) gene on a plasmid vector. Expression of the cat gene was subject to repression by tyrR+. This fusion was used to isolate regulatory mutants with increased expression of the cat gene in which repression by tyrR+ was affected. Nucleotide sequencing of these mutants has led to the identification of three sites involved in the repression of aroF by tyrR+. The existence of a functional promoter divergently transcribing from the aroF regulatory region was also demonstrated by using the cat fusion vector. The expression of this promoter is also regulated by tyrR+.     

Regulation of tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: properties of the tyrR gene product

A spontaneous amber tyrR mutant has been isolated in which constitutive synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and DAHP synthetase (phe) is suppressible by supC(-), supD(-), supF(-) and supU(-). This finding suggests the tyrR gene product is a protein. Derepression of DAHP synthetase (phe) in this and in seven other spontaneous tyrR mutants and in four Mu-1-induced tyrR mutants provides further evidence for the involvement of the tyrR gene product in phenylalanine biosynthesis. Evidence that the tyrR product is a component of repressor, rather than an enzyme involved in its synthesis or modification, comes from a study of a temperature-sensitive tyrR mutant. This mutant is of the thermolabile type, since derepression occurs rapidly and in the presence and absence of growth.     

Formation of a lambda (Tn10) tyrR+ specialized transducing bacteriophage from Escherichia coli K-12.

The transposon Tn10, coding for resistance to tetracycline, was inserted close to the tyrR+ gene at min 28 on the Escherichia coli chromosome. The homology between this transposon and a lambda (Tn10) phage was employed to direct integration of lambda close to tyrR+ with subsequent isolation of a lambda (Tn10) tyrR+ transducing phage. Results of restriction endonuclease analysis of the transducing phage are presented.     

大肠杆菌tyrR基因剔除及其对苯丙氨酸生物合成的影响

TyrR是大肠杆菌芳香族氨基酸生物合成和运输途径中的一种全局性调控蛋白质。采用双交换同源重组的方法定位突变大肠杆菌染色体tyrR基因 ,在该基因中插入带有卡那霉素抗性基因的DNA片段 ,使之失活 ,实现基因剔除。经PCR、DNA测序、lacZ报告基因等多种方法证实了基因剔除的可靠性。tyrR基因剔除后 ,大肠杆菌芳香族氨基酸生物合成中受TyrR蛋白调控的关键酶的酶活力有所提高 :3 脱氧 2 阿拉伯庚酮糖 7 磷酸合成酶(DAHPS ,由aroG编码 )酶活力提高了 1.0 8倍 ,转氨酶 (AT ,由tyrB编码 )酶活力提高了 2 .70倍 ;突变菌株发酵生产苯丙氨酸的能力提高了 1.5 9倍 ;同时 ,与芳香族氨基酸运输相关的通透酶基因aroP(P)的阻遏被解除 ,细胞运输芳香族氨基酸的能力提高了 70 .2 %。     

Repression of aromatic amino acid biosynthesis in Escherichia coli K-12

Mutants of Escherichia coli K-12 were isolated in which the synthesis of the following, normally repressible enzymes of aromatic biosynthesis was constitutive: 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases (phe and tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. In the wild type, DAHP synthetase (phe) was multivalently repressed by phenylalanine plus tryptophan, whereas DAHP synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A were repressed by tyrosine. DAHP synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase were also repressed by phenylalanine in high concentration (10(-3)m). Besides the constitutive synthesis of DAHP synthetase (phe), the mutants had the same phenotype as strains mutated in the tyrosine regulatory gene tyrR. The mutations causing this phenotype were cotransducible with trpA, trpE, cysB, and pyrF and mapped in the same region as tyrR at approximately 26 min on the chromosome. It is concluded that these mutations may be alleles of the tyrR gene and that synthesis of the enzymes listed above is controlled by this gene. Chorismate mutase P and prephenate dehydratase activities which are carried on a single protein were repressed by phenylalanine alone and were not controlled by tyrR. Formation of this protein is presumed to be controlled by a separate, unknown regulator gene. The heat-stable phenylalanine transaminase and two enzymes of the common aromatic pathway, 5-dehydroquinate synthetase and 5-dehydroquinase, were not repressible under the conditions studied and were not affected by tyrR. DAHP synthetase (trp) and tryptophan synthetase were repressed by tryptophan and have previously been shown to be under the control of the trpR regulatory gene. These enzymes also were unaffected by tyrR.     

Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A

Mutant strains of Escherichia coli have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (phe) is derepressed, in addition to those enzymes of tyrosine biosynthesis previously shown to be controlled by the gene tyrR. The major enzyme of the terminal pathway of phenylalanine biosynthesis chorismate mutase-prephenate dehydratase is not derepressed in these strains. Genetic analysis of the mutants shows that the mutation or mutations causing derepression map close to previously reported tyrR mutations. A study of one of the mutations has shown it to be recessive to the wild-type allele in a diploid strain. It is proposed that the tyrR gene product is involved in the regulation of the synthesis of DAHP synthetase (phe) as well as the synthesis of DAHP synthetase (tyr), chorismate mutase-prephenate dehydrogenase, and transaminase A.     

The tyrosine repressor negatively regulates aroH expression in Escherichia coli.

The levels of the tryptophan-sensitive isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli, encoded by the aroH gene, were elevated in tyrR and/or trpR mutants. The effect of tyrR and trpR lesions on aroH expression was confirmed by using a lacZ reporter system. The mutational elimination of either repressor led to a threefold increase in beta-galactosidase.     

Regulation of tyrosine and phenylalanine biosynthesis in Salmonella.

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.     

Tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: complementation between different tyrR alleles

Dominance tests in diploids have confirmed that the product of the tyrR gene is involved in a negative control system affecting the synthesis of both 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and DAHP synthetase (phe). Some tyrR mutants are derepressed for the synthesis of both DAHP synthetase (tyr) and (phe), whereas others are derepressed for the synthesis of DAHP synthetase (tyr) but overrepressed for the synthesis of DAHP synthetase (phe). Complementation tests between these alleles confirm that they are in the same cistron. The allele causing overrepression of enzyme synthesis is dominant over both the wild type and the derepressing allele in diploids.     

Regulation of aromatic amino acid transport systems in Escherichia coli K-12.

The regulation of the aromatic amino acid transport systems was investigated. The common (general) aromatic transport system and the tyrosine-specific transport system were found to be subject to repression control, thus confirming earlier reports. In addition, tryosine- and tryptophan-specific transport were found to be enhanced by growth of cells with phenylalanine. The repression and enhancement of the transport systems was abolished in a strain carrying an amber mutation in the regulator gene tyrR. This indicates that the tyrR gene product, which was previously shown to be involved in regulation of aromatic biosynthetic enzymes, is also involved in the regulation of the aromatic amino acid transport systems.     

Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.     

Isolation and analysis of aroFo mutants by using an aroF-lac operon fusion

The lac structural genes were fused to the regulatory region of the aroF-tyrA operon so that the expression of beta-galactosidase was regulated by the tyrR+ gene product. Transducing phage carrying the aroF-lac fusion were isolated, and a lambda aroF-lac lysogen was used to select for aroFo mutants. A plasmid vector was constructed onto which the aroFo mutations were transferred by recombination in vivo.     

Regulator gene controlling enzymes concerned in tyrosine biosynthesis in Escherichia coli

Mutants of Escherichia coli K-12 have been isolated in which several enzymes concerned with tyrosine biosynthesis are derepressed. These mutants were obtained from a parent strain possessing only a single 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase isoenzyme, DAHP synthetase (tyr), by selecting for resistance to the tyrosine analogue, 4-aminophenylalanine. The mutation responsible for this derepression has been mapped and the gene, which is not closely linked to aroF and tyrA, has been designated tyrR.     

Instability in tyrR Strains of Plasmids Carrying the Tyrosine Operon: Isolation and Characterization of Plasmid Derivatives with Insertions or Deletions

The transformation of tyrR strains of Escherichia coli with multicopy plasmids which carry the tyrosine operon gave rise to modified plasmids with either insertions or deletions. The effect of each of these insertions or deletions was to decrease the level of expression of this operon. It is proposed that plasmid instability arose as a direct consequence of the metabolic effects of an overproduction of the enzymes coded for by the tyrosine operon. The results have significant implications for the cloning of genes that are repressed by the product of a regulatory gene. Since the predominant plasmid modification observed was the insertion of an IS1 element near the regulatory region of the tyrosine operon, the results also suggest a role for IS1 elements in the regulation of gene expression.