Role of the Escherichia coli aromatic amino acid aminotransferase in leucine biosynthesis.

Strains of Escherichia coli that lack the branched-chain amino acid amino-transferase because of mutations in the ilvE gene had no growth requirement for leucine when the cells contained the aromatic amino acid aminotransferase that is the product of the tyrB gene. The presence of leucine increased the generation time of these cells and decreased the specific activity of the aromatic amino acid aminotransferase. It is concluded that this enzyme functions efficiently in leucine biosynthesis and can be repressed by leucine as well as by tyrosine.     

A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli

A transposon-induced mutation in Escherichia coli resulted in a lack of two modified nucleosides in the transfer ribonucleic acid. These nucleosides were identified as uridine-5-oxyacetic acid (cmo⁵U)² and its methylester, mcmo⁵U. Both became radioactively labelled using [methyl-¹⁴C]methionine as methyl donor when wild-type cells were grown in a defined rich medium. We believe that both nucleosides have hydroxyuridine as a common precursor, which should be methylated in the first modification step. However, in our system in vitro the tRNA from the mutant was not a methyl group acceptor, indicating that the step affected in the mutant occurs before the methylation step. Thus, the most likely biosynthetic pathway is: formation of (1) hydroxyuridine, (2) methoxyuridine. (3) cmo⁵U and, in some cases, (4) mcmo⁵U. The mutant had also become Aro^?, i.e. it required aromatic amino acids for growth. Genetic analysis revealed that the transposon Tn5 had been integrated close to or within the aroD gene, the gene product of which participates in the synthesis of shikimic acid. The common pathway of the biosynthesis of aromatic amino acids includes the genes aroB, D, E, A and C in that order, and any mutant defective in any of these genes lacked cmo⁵U and mcmo⁵U in their tRNA. When shikimic acid was included in the defined rich medium used, the Tn5-induced mutant regained the normal level of cmo⁵U and mcmo⁵U while an aroC mutant (distal to shikimic acid but prior to chorismic acid) did not. The rich medium used contained, besides the aromatic amino acids, all the precursors for the synthesis of folate, ubiquinone and enterochelin. Thus, chorismic acid itself or a metabolite of it in the synthetic pathway to vitamin K₂ or in an unknown pathway must play a pivotal role in this specific modification of the tRNA. These results reveal a novel link between the biosynthesis of amino acids and modification of tRNA.     

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.     

YddG from Escherichia coli promotes export of aromatic amino acids

The inner membrane protein YddG of Escherichia coli is a homologue of the known amino acid exporters RhtA and YdeD. It was found that the yddG gene overexpression conferred resistance upon E. coli cells to the inhibiting concentrations of l-phenylalanine and aromatic amino acid analogues, dl-p-fluorophenylalanine, dl-o-fluorophenylalanine and dl-5-fluorotryptophan. In addition, yddG overexpression enhanced the production of l-phenylalanine, l-tyrosine or l-tryptophan by the respective E. coli-producing strains. On the other hand, the inactivation of yddG decreased the aromatic amino acid accumulation by these strains. The cells of the E. colil-phenylalanine-producing strain containing overexpressed yddG accumulated less l-phenylalanine inside and exported the amino acid at a higher rate than the cells of the isogenic strain containing wild-type yddG. Taken together, these results indicate that YddG functions as an aromatic amino acid exporter.     

Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.

Two aminotransferases from Escherichia coli were purified to homogeneity by the criterion of gel electrophoresis. The first (enzyme A) is active on L-aspartic acid, L-tyrosine, L-phenylalanine, and L-tryptophan; the second (enzyme B) is active on the aromatic amiono acids. Enzyme A is identical in substrate specificity with transaminase A and is mainly an aspartate aminotransferase; enzyme B has never been described before and is an aromatic amino acid aminotransferase. The two enzymes are different in the Vmax and Km values with their common substrates and pyridoxal phosphate, in heat stability (enzyme A being heat-stable and enzyme B being heat-labile at 55 degrees) and in pH optima with the amino acid substrates. They are similar in their amino acid composition, each enzyme appears to consist of two subunits, and enzyme B may be converted to enzyme A by controlled proteolysis with subtilsin. The conversion was detected by the generation of new aspartate aminotransferase activity from enzyme B and was further verified by identification by acrylamide gel electrophoresis of the newly formed enzyme A. The two enzymes appear to be products of two genes different in a small, probably terminal, nucleotide sequence.     

Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli.

The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis.     

Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria.

We examined the enzymology and regulatory patterns of the aromatic amino acid pathway in 48 strains of cyanobacteria including representatives from each of the five major grouping. Extensive diversity was found in allosteric inhibition patterns of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, not only between the major groupings but also within several of the generic groupings. Unimetabolite inhibition by phenylalanine occurred in approximately half of the strains examined; in the other strains unimetabolite inhibition by tyrosine and cumulative, concerted, and additive patterns were found. The additive patterns suggest the presence of regulatory isozymes. Even though both arogenate and prephenate dehydrogenase activities were found in some strains, it seems clear that the arogenate pathway to tyrosine is a common trait that has been highly conserved among cyanobacteria. No arogenate dehydratase activities were found. In general, prephenate dehydratase activities were activated by tyrosine and inhibited by phenylalanine. Chorismate mutase, arogenate dehydrogenase, and shikimate dehydrogenase were nearly always unregulated. Most strains preferred NADP as the cofactor for the dehydrogenase activities. The diversity in the allosteric inhibition patterns for 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, cofactor specificities, and the presence or absence of prephenate dehydrogenase activity allowed the separation of subgroupings within several of the form genera, namely, Synechococcus, Synechocystis, Anabaena, Nostoc, and Calothrix.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.

Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli. One mutation, designated tyrB, lies at about 80 min on the E. coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase. The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids. In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis. The ilvE gene product alone can reverse a phenylalanine requirement. Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate. These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility. In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.     

A new family of integral membrane proteins involved in transport of aromatic amino acids in Escherichia coli.

The nucleotide sequence of tnaB of the tryptophanase operon of Escherichia coli is presented. TnaB is a tryptophan-specific permease that is homologous to Mtr, a second tryptophan-specific permease, and to TyrP, a tyrosine-specific permease. Each member of this family appears to contain 11 membrane-spanning domains.     

Maintenance and exchange of the aromatic amino acid pool in Escherichia coli

The pool of phenylalanine, tyrosine, and tryptophan is formed in Escherichia coli K-12 by a general aromatic transport system [Michaelis constant (K(m)) for each amino acid approximately 5 x 10(-7)m] and three further transport systems each specific for a single aromatic amino acid (K(m) for each amino acid approximately 2 x 10(-6)m, reference 3). When the external concentration of a particular aromatic amino acid is saturating for both classes of transport system, the free amino acid pool is supplied with external amino acid by both systems. Blocking the general transport system reduces the pool size by 80 to 90% but does not interfere with the supply of the amino acid to protein synthesis. If, however, the external concentration is too low to saturate specific transport, blocking general transport inhibits the incorporation of external amino acid into protein by about 75%. It is concluded that the amino acids transported by either class of transport system can be used for protein synthesis. Dilution of the external amino acid or deprivation of energy causes efflux of the aromatic pool. These results and rapid exchange observed between pool amino acid and external amino acids indicate that the aromatic pool circulates rapidly between the inside and the outside of the cell. Evidence is presented that this exchange is mediated by the aromatic transport systems. Mutation of aroP (a gene specifying general aromatic transport) inhibits exit and exchange of the small pool generated by specific transport. These findings are discussed and a simple physiological model of aromatic pool formation, and exchange, is proposed.     

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.     

Distribution and function of genes concerned with aromatic biosynthesis in Escherichia coli

Pittard, James (School of Microbiology, University of Melbourne, Victoria, Australia), and B. J. Wallace. Distribution and function of genes concerned with aromatic biosynthesis in Escherichia coli. J. Bacteriol. 91:1494-1508. 1966.-A number of mutant strains of Escherichia coli K-12, which are blocked in the biosynthesis of the aromatic amino acids, were examined biochemically to determine their particular enzymatic deficiencies. The mutations carried by these strains were mapped by use of the methods of conjugation and transduction. Structural genes for five of the enzymes of the common pathway leading to chorismate and for the two enzymes converting chorismate to phenylpyruvate and p-hydroxyphenylpyruvate, respectively, were identified. Unlike the genes of the tryptophan operon most of these genes are distributed over widely separated regions of the chromosome.