Neurospora Mutant Deficient in Tryptophanyl-Transfer Ribonucleic Acid Synthetase Activity

A tryptophan auxotroph of Neurospora crassa, trp-5, has been characterized as a mutant with a deficient tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.2) activity. When assayed by tryptophanyl-tRNA formation, extracts of the mutant have less than 5% of the wild-type specific activity. The adenosine triphosphate-pyrophosphate exchange activity is at about half the normal level. In the mutant derepressed levels of anthranilate synthetase and tryptophan synthetase were associated with free tryptophan pools equal to or higher than those found in the wild type. We conclude that a product of the normal tryptophanyl-tRNA synthetase, probably tryptophanyl-tRNA, rather than free tryptophan, participates in the repression of the tryptophan biosynthetic enzymes.     

Temperature-induced derepression of tryptophan biosynthesis in a tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (l-tryptophan: tRNA ligase adenosine monophosphate, EC 6.1.1.2) mutant (trpS1) of Bacillus subtilis is derepressed for enzymes of the tryptophan biosynthetic pathway at temperatures which reduce the growth rate but still allow exponential growth. Derepression of anthranilate synthase in a tryptophan-supplemented medium (50 mug/ml) is maximal at 36 C, and the differential rate of synthesis is 600- to 2,000-fold greater than that of the wild-type strain or trpS1 revertants. A study of the derepression pattern in the mutant and its revertants indicates that the 5-fluorotryptophan recognition site of the tryptophanyl-tRNA synthetase is an integral part of the repression mechanism. Evidence for a second locus, unlinked to the trpS1 locus, which functions in the repression of tryptophan biosynthetic enzymes is presented.     

Regulation of E. coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNA^Phe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

Regulation of the tryptophan synthetic enzymes in Clostridium butyricum

Experiments concerned with the regulation of the tryptophan synthetic enzymes in anaerobes were carried out with a strain of Clostridium butyricum. Enzyme activities for four of the five synthetic reactions were readily detected in wild-type cells grown in minimal medium. The enzymes mediating reactions 3, 4, and 5 were derepressed 4- to 20-fold, and the data suggest that these enzymes are coordinately controlled in this anaerobe. The first enzyme of the pathway, anthranilate synthetase, could be derepressed approximately 90-fold under these conditions, suggesting that this enzyme is semicoordinately controlled. Mutants resistant to 5-methyl tryptophan were isolated, and two of these were selected for further analysis. Both mutants retained high constitutive levels of the tryptophan synthetic enzymes even in the presence of repressing concentrations of tryptophan. The anthranilate synthetase from one mutant was more sensitive to feedback inhibition by tryptophan than the enzyme from wild-type cells. The enzyme from the second mutant was comparatively resistant to feedback inhibition by tryptophan. Neither strain excreted tryptophan into the culture fluid. Tryptophan inhibits anthranilate synthetase from wild-type cells noncompetitively with respect to chorismate and uncompetitively with respect to glutamine. The Michaelis constants calculated for chorismate and glutamine are 7.6 x 10(-5)m and 6.7 x 10(-5)m, respectively. The molecular weights of the enzymes estimated by zonal centrifugation in sucrose and by gel filtration ranged from 24,000 to 89,000. With the possible exception of a tryptophan synthetase complex, there was no evidence for the existence of other enzyme aggregates. The data indicate that tryptophan synthesis is regulated by repression control of the relevant enzymes and by feedback inhibition of anthranilate synthetase. That this enzyme system more closely resembles that found in Bacillus than that found in enteric bacteria is discussed.     

Evidence That the Majority of Leucine Transfer Ribonucleic Acid Is Not Involved in Repression in Salmonella typhimurium

Leucine transfer ribonucleic acid (tRNA) was almost fully charged, and the isoleucine-valine and leucine enzymes remained derepressed when trifluoroleucine was added to a leucine auxotroph. High levels of charged leucine tRNA and derepression were also found in a leucyl-tRNA synthetase mutant.     

Multivalent repression and genetic depression of isoleucine-valine biosynthetic enzymes in Serratia marcescens

The regulation of the formation of isoleucine-valine biosynthetic enzymes was examined to elucidate the mechanism of isoleucine-valine accumulation by alpha-aminobutyric acid-resistant (abu-r) mutants of Serratia marcescens. In the isoleucine-valine auxotroph, l-threonine dehydratase, acetohydroxy acid synthetase, and transaminase B were repressed when isoleucine, valine, and leucine were simultaneously added to minimal medium. These enzymes were derepressed at the limitation of any single branched-chain amino acid. Pantothenate, which stimulated growth of this auxotroph, had no effect on the enzyme levels. It became evident from these results that in S. marcescens isoleucine-valine biosynthetic enzymes are subject to multivalent repression by three branched-chain amino acids. The abu-r mutants had high enzyme levels in minimal medium, with or without three branched-chain amino acids. Therefore, in abu-r mutants, isoleucine-valine biosynthetic enzymes are genetically derepressed. This derepression was considered to be the primary cause for valine accumulation and increased isoleucine accumulation.     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.     

Histidine-Mediated Control of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of the five tryptophan biosynthetic enzymes of Neurospora crassa was shown to be derepressed in histidine-starved cells. This histidine-mediated derepression was not due to a lowered intracellular concentration of tryptophan in these cells. Furthermore, histidine-mediated derepression of tryptophan enzymes was found to be coordinate and not subject to reversal by tryptophan of either exogenous or biosynthetic origin. The synthesis of tryptophan enzymes also was found to be coordinate in cells which were not histidine-starved. Although histidine is clearly involved in regulating the synthesis of tryptophan enzymes, it did not prevent either tryptophan-mediated derepression of tryptophan enzymes or indole-3-glycerol phosphate-mediated derepression of tryptophan synthetase.     

Synthesis of l-Tryptophan from Indole and dl-Serine by Tryptophan Synthetase Entrapped in Fibres

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.

The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.

The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.

Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 days) provided the accumulation of indole on the fibres was avoided.     

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.     

Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

Biosynthesis and Characterization of 4-Fluorotryptophan-Labeled Escherichia coli Arginyl-tRNA Synthetase

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher K _m values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of ¹⁹F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.     

Studies on tryptophan synthetases from various strains of Bacillus subtilis

1. Tryptophan synthetase B of three strains of Bacillus subtilis was prepared from ;exo-protoplastic' and ;endo-protoplastic' fractions; the enzyme from ;exo-protoplastic' fraction was purified 30- to 120-fold by ammonium sulphate precipitation and DEAE-cellulose column chromatography; the latter step separated this enzyme from tryptophan synthetase A, tryptophanase and proteolytic enzymes, but the purified preparations were not stable. 2. The activity of tryptophan synthetase B did not depend on the presence of tryptophan synthetase A. 3. Tryptophan synthetases B of the strains tested differed in their utilization of 2- and 7-methylindole as compared with indole; this suggests that these tryptophan synthetases B are not identical.     

Role of Isoleucyl-Transfer Ribonucleic Acid Synthetase in Ribonucleic Acid Synthesis and Enzyme Repression in Yeast

Temperature-sensitive mutations in the isoleucyl-transfer ribonucleic acid (tRNA) synthetase of yeast, ilS(-)1-1 and ilS(-)1-2, were used to examine the role of aminoacyl-tRNA synthetase enzymes in the regulation of ribonucleic acid (RNA) synthesis and enzyme synthesis in a eucaryotic organism. At the permissive temperature, 70 to 100% of the intracellular isoleucyl-tRNA was charged in mutants carrying these mutations; at growth-limiting temperatures, less than 10% was charged with isoleucine. Other aminoacyl-tRNA molecules remained essentially fully charged under both conditions. Net protein and RNA syntheses were rapidly inhibited when the mutant was shifted from the permissive to the restrictive temperature. Most of the ribosomes remained in polyribosome structures at the restrictive temperature even though protein synthesis was strongly inhibited. Two of the enzymes of isoleucine biosynthesis, threonine deaminase and acetohydroxyacid synthetase, were derepressed about twofold during slow growth of the mutants at a growth-limiting temperature. This is about the same degree of derepression that is achieved by growth of an auxotroph on limiting isoleucine. We conclude that charged aminoacyl-tRNA is essential for RNA synthesis and for the multivalent repression of the isoleucine biosynthetic enzymes. Aminoacyl tRNA synthetase enzymes appear to play important regulatory roles in the cell physiology of eucaryotic organisms.     

Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus

The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.     

On the role of isoleucyl-tRNA synthetase in multivalent repression

An isoleucine auxotroph of Salmonella typhimurium was derived from a merodiploid strain (containing the F-14 episome from Escherichia coli) that contained two copies of the structural genes concerned with isoleucine and valine biosynthesis. A haploid derivative, strain TU6001, having the same growth properties as the original merodiploid mutant was found to have normal biosynthetic enzymes and an altered isoleucyl-tRNA synthetase. The K _m for isoleucine was increased by about 200-fold over that for the wild-type enzyme. All five enzymes in the isoleucine and valine biosynthetic pathway were derepressed relative to wild-type enzyme levels. A partial revertant of strain TU6001 was isolated which had properties that were intermediate between those of the mutant and the wild type (i.e., intermediate growth dependence on exogenous isoleucine, intermediate activity of isoleucyl-tRNA synthetase, and intermediate derepression of biosynthetic enzymes). The properties of strain TU6001 were demonstrated to be simultaneously transferable by transduction (using P_LT22 H₄ bacteriophage) of a single genetic locus, linked to pyr A, which has been designated ilv S. It is concluded that some function of the isoleucyl-tRNA synthetase is important in repression of the isoleucine and valine biosynthetic enzymes.Supported by grant GM 12522 from the National Institute of General Medical Sciences, U.S. Public Health Service. J. M. B. received a U.S. Public Health Service Postdoctoral Fellowship 1-F02-GM-30, 650-02.     

Influences of Various Amino Acids on Tryptophan-Mediated Control of the Tryptophan Biosynthetic Enzymes in Escherichia coli

Lysates of Escherichia coli Ymel obtained from cultures grown in the absence of tryptophan in minimal medium supplemented with 0.1% casein hydrolysate show an approximate fivefold increase in steady-state specific activity of both anthranilate synthetase and tryptophan synthetase A protein relative to cultures grown in nonsupplemented medium. In the presence of repressing levels of exogenous tryptophan, growth of cultures in casein hydrolysate-supplemented medium results in a noncoordinate enhancement of repression of 10-fold for anthranilate synthetase and twofold for tryptophan synthetase A protein. Similar, but less pronounced, effects are shown for strain W3110. Strains possessing tryptophan regulator gene mutations do not exhibit this first effect, but do yield an approximate twofold decrease in specific activity of both enzymes when grown in medium supplemented with tryptophan and casein hydrolysate. A stimulation of derepression of both enzymes in strain Ymel equivalent to that induced by casein hydrolysate can be reproduced by growth in minimal medium supplemented with threonine, phenylalanine, tyrosine, serine, glutamic acid, and glutamine. Doubling time in this medium is not significantly different from that in minimal medium. An enhancement of repression which partially mimics that observed on growth in medium supplemented with tryptophan plus casein hydrolysate is obtained when Ymel is grown on medium supplemented with tryptophan plus methionine. Threonine or phenylalanine plus tyrosine as separate medium supplements are independently capable of producing a 1.4-fold or 3.4-fold stimulation, respectively, but in combination only the phenylalanine plus tyrosine effect is manifested unless serine and glutamic acid or glutamine are included. Our data show that expression of the tryptophan biosynthetic enzymes can be significantly influenced in vivo as a result of growth in medium supplemented with a variety of amino acids.     

Enzymatic production of tryptophan using a lipoic acid and thiamine double auxotroph of Enterobacter aerogenes having both pyruvic acid productivity and high tryptophanase activity

Washed cells of a newly-developed double auxotroph for lipoic acid and thiamine, Enterobacter aerogenes LT-94, produced about 30 g/l of pyruvic acid from 5% glucose, and subsequently converted the acid to tryptophan in a yield of 16.7 g/l by the reverse reaction of tryptophanase, which had been induced under derepressed conditions. These yields were higher than those of the parent strain, L-12, showing only lipoic acid auxotrophy.     

Preferential utilization of glutamine for amination of xanthosine 5'-phosphate to guanosine 5'-phosphate by purified enzymes from Escherichia coli

GMP synthetase was purified 180-fold from $\text{E. coli}$ B and 18-fold from the derepressed purine auxotroph, $\text{E. coli}$ B-96. The enzymes from both sources show the same preference for glutamine over ammonia as amino donor. Each is dimeric, consisting of subunits of molecular weight about 60,000. Thus the two are apparently identical. The similarities between GMP synthetase and xanthosine 5′-phosphate aminase of $\text{E. coli}$ B-96 (N. Sakamoto, G.W. Hatfield, and H.S. Moyed, J. Biol. Chem. (1972) $\text{247}$, 5880–5887) in respect to structure, state of derepression, and behavior during purification, lead us to the conclusion that the synthetase and the aminase are a single entity. We observe no loss or separation of glutamine-dependent activity upon purification of GMP synthetase and we suggest that such loss, reported by other workers, results artifactually by inactivation of an intrinsic glutamine-binding site. GMP synthetase appears not to contain a glutamine-binding subunit which is separable from the xanthosine 5′-phosphate-aminating component.     

The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.