Thialysine- and selenalysine-resistance in a E. coli mutant

A thialysine-resistant mutant of E. coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium. No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain.     

Biochemical characterization of a thialysine-resistant clone of CHO cells

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.     

Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine.

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition casued by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzyme synthesis as well as inhibition of activity.     

Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition caused by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzymes synthesis as well as inhibition of activity.     

Genetic and biochemical properties of thialysine-resistant mutants of Saccharomyces cerevisiae

Three groups of lysine-excreting, thialysine-resistant mutants of Saccharomyces cerevisiae were derived from the wild-type strain (X2180) by mutagenic treatment and selected on the basis of a cross-feeding assay. Mutants MNNG2-9, MNNG2-27, MNNG2-39 and MNNG2-62 (group 1) exhibited a 2:2 segregation for thialysine resistance following mating with a wild-type strain and a lower than wild-type lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was dominant in specific hybrids. Mutant MNNG2-2 (group II) was similar to group I mutants except that the thialysine-resistant phenotype was recessive in the hybrid. Mutant MNNG3-142 (group III) exhibited an irregular ratio of segregation of thialysine resistance and a significantly lower lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was recessive in the hybrid. The growth of both group I and group III mutants was temperature-sensitive. The thialysine-resistant mutants exhibited pleiotropic properties including the increased production and excretion of lysine, thermosensitive growth and an impairment of lysyl-tRNA synthetase activity.     

Effect of mutations affecting lysyl-tRNAlys on the regulation of lysine biosynthesis in Escherichia coli.

Summary When studying mutants affecting lysyl-tRNA synthetase or tRNA^Lys (hisT, hisW), a lack of correlation is clearly observed between the amount of lysyl-tRNA and the level of derepression of several lysine biosynthetic enzymes. This excludes the possible role of lysyl-tRNA as the specific corepressor of the lysine regulon. However, the level of derepression of DAP-decarboxylase, the last enzyme of the lysine pathway, is very low in the hisT mutant; this indicates that tRNA^Lys is a secondary effector involved in the regulation of the synthesis of this enzyme.Abbreviations DAP diaminopimelate - KRS lysyl-tRNA synthetase - L-lysine tRNA ligase (AMP) (EC6.1.16) - AK III lysinesensitive aspartokinase (EC 2.7.24) - ASA-dehydrogenase aspartic semialdehyde dehydrogenase (EC 1.2.1.10) - DHDP-reductase dihydrodipicolinic acid reductase - DAP-decarboxylase diaminopimelate decarboxylase (EC 4.1.1.20) - AK I threonine-sensitive aspartokinase - HDHI threonine-sensitive homoserine dehydrogenase     

Role of lysyl-tRNA in the regulation of lysine biosynthesis in Escherichia coli K12.

A mutant of lysyl-tRNA synthetase has been isolated in Escherichia coli K12. With this strain the Kmapp for lysine is 25 fold higher than with the parental strain. The percentage of charged tRNAlys in vivo is only 7 per cent (as against 65 per cent with HFR H). Under these conditions no derepression of synthesis is observed for three lysine biosynthetic enzymes (AK III, ASA-dehydrogenase, DAP-decarboxylase) ; a partial derepression is obtained in the case of the dhdp-reductase. Thus lysyl-tRNA does not act as the only corepressor molecule in the lysine regulon.     

Aspartokinase III repression and lysine analogs utilization for protein synthesis

The extents of thialysine and selenalysine incorporation into cell proteins were compared in E. coli KL16 and in a mutant able to grow equally well in the presence or in the absence of both lysine analogs. The mutant differs from the parental strain in the repression of aspartokinase III (AKIII), the first enzyme of the lysine biosynthetic pathway. No analog incorporation into proteins was observed in mutant cells grown in the presence of either analog, whereas a marked analog incorporation was observed in the parental strain, where up to 17% and 12% of protein lysine can be substituted by thialysine and selenalysine respectively. In the parental strain grown in media containing either analog at different concentration the extent of analog incorporation into proteins is related to the extent of AKIII repression.     

Two modes of metabolic regulation of lysyl-transfer ribonucleic acid synthetase in Escherichia coli K-12.

Lysyl-transfer ribonucleic acid (tRNA) synthetase activity was compared in three independently isolated Escherichia coli K-12 mutants of the enzyme S-adenosyl-L-methionine synthetase (metK mutants) and their isogenic parents. In all three cases the activity of the lysyl-tRNA synthetase was elevated two- to fourfold in the mutant strains. Glycyl-L-leucine (3 mM) usually enhanced lysyl-tRNA synthetase activity two- to threefold in wild-type cells but did not further stimulate the synthetase activity in metK mutants. By two other criteria, the lysyl-tRNA synthetase from wild-type cells grown with the peptide and from the metK mutant RG62, grown in minimal medium, were similar. These criteria are enhanced resistance to thermal inactivation and altered susceptibility to endogenous proteases when compared with the synthetase from wild-type cells grown in minimal medium. In a separate set of experiments, the activities of the lysyl-, arginyl-, seryl-, and valyl-tRNA synthetases were measured in an isogenic pair of relt and rel strains of E. coli grown in a relatively poor growth medium (acetate) and in enriched medium. In the rel+ strain the level of all four synthetases was higher (two- to fourfold) in the enriched medium as expected. In the rel strain the difference in the activities of the synthetases between the two media were diminished. In all four cases the activities of the synthetases were higher in acetate medium in the rel strain. Evidence is presented that these two modes of metabolic regulation act independently.     

Thialysine utilization for protein synthesis by an exponentially growing E. coli culture

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.     

Structure and evolution of a group of related aminoacyl-tRNA synthetases

A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.     

Repression of lysine transport in E. coli KL16

Data reported in this paper show that both lysine transport systems in E. coli KL16 can be repressed by lysine and its isologs, thialysine and selenalysine, whereas they are not repressed by ornithine. The repression is specific on lysine transport systems; it is evident with 0.01 mM lysine or isolog concentration and reaches a maximum with 0.1 mM concentration. By comparing the extent of repression by lysine and its isologs, lysine gives the highest and selenalysine the lowest degree of repression. The shift from the repressed to the depressed state is rather immediate once the amino acid is removed from the culture medium.     

Escherichia coli K-12 lysyl-tRNA synthetase mutant with a novel reversion pattern

Fast-growing revertants have been selected from a slow-growing lysyl-tRNA synthetase mutant. All of the revertants had increased lysyl-tRNA synthetase activity compared with the mutant (5- to 85-fold), and in some revertants this amounted to two to three times the wild-type synthetase activity. Two-dimensional gel electrophoresis of a whole-cell extract of revertant IH2018 (1.5- to 2-fold wild-type synthetase activity) showed that the increase in synthetase activity is due to the induction of cryptic lysyl-tRNA synthetase forms and not to a change in the constitutive lysyl-tRNA synthetase. Genetic studies have shown that a locus termed rlu (for regulation of lysU ) which is cotransducible with purF at 49.5 min influences the amount of the cryptic lysyl-tRNA synthetase.     

Defects of two temperature-sensitive lysyl-transfer ribonucleic acid synthetase mutants of Bacillus subtilis

Two temperature-sensitive mutants (lysS1 and lysS2) of the lysyl-transfer ribonucleic acid synthetase (l-lysine:tRNA ligase [adenosine 5'-monophosphate], EC 6.1.1.6) of Bacillus subtilis have been isolated. Although protein synthesis is inhibited in both mutants at the restrictive temperature (42 to 45 C), the mutants remain viable in a minimal medium. In comparison with the wild-type lysyl-tRNA synthetase, the l-lysine-dependent exchange of [(32)P]pyrophosphate with adenosine 5'-triphosphate (ATP) for both mutant enzymes is decreased. The lysS1 enzyme is completely defective in the ATP-dependent attachment of l-lysine to tRNA, whereas the lysS2 enzyme has 3- to 10-fold reduced levels of this activity. Temperature-resistant transformants have wild-type enzyme levels, whereas partial revertants to temperature resistance have varied levels of enzyme activity. The attachment and exchange activities of the lysS2 enzyme are more heat labile in vitro than the wild-type enzyme, as is the attachment activity of a partial revertant of the lysS1 mutant. The lysS1 and the lysS2 lysyl-tRNA synthetases have higher apparent K(m) values for lysine and ATP, in both the activation and the attachment reactions. The lysS2 enzyme has a V(max) for tRNA(lys) one-third that of the wild-type enzyme. Molecular weights of approximately 150,000 for the wild-type and lysS2 enzymes and approximately 76,000 for the lysS1 enzyme were estimated from sedimentation positions in sucrose density gradients assayed by the ATP-pyrophosphate exchange activity. We propose that the two mutations (lysS1 and lysS2) directly affect the sites for exchange activity, but indirectly alter attachment activity as a consequence of defective subunit association.     

Independent regulation of transport and biosynthesis of arginine in Escherichia coli K-12.

From an arginine auxotrophic strain, a mutant was isolated which is able to utilize d-arginine as a source of l-arginine and shows a high sensitivity to inhibition of growth by canavanine. Transport studies revealed a four- to five-fold increased uptake of arginine and ornithine in cells from the mutant strain. The kinetics of entry of arginine and ornithine evidenced elevated maximal influx values for the arginine- and ornithine-specific transport systems. A close parallel between arginine transport activity and arginine binding activity with one arginine-specific binding periplasmic protein in the mutant strongly suggests that such binding protein is a component of the arginine-specific permease. The affinity between arginine and the binder, isolated from the mutant cells, as well as the electrophoretic mobility of the protein, remain unchanged. The enhanced transport activity of arginine and ornithine with mutant cells is insensitive to repression by arginine or ornithine, whereas the biosynthesis of arginine-forming enzymes is normally repressible. When transport activity was examined in strains with mutations leading to derepression of arginine biosynthesis, the regulation of arginine transport was found to be normal. These studies support the conclusion that arginine transport and arginine biosynthesis, in Escherichia coli K-12, are not regulated in a concerted manner, although both systems may have components in common.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Streptococcus faecalis

We were able to show that two lysine-independent mutants of Streptococcus faecalis ATCC 8043 contained the enzymes for the usual bacterial pathway for lysine biosynthesis. Because of this synthetic capacity, one mutant, the Lys(+)OHLys(s) strain, could not grow in the presence of hydroxylysine without a lysine supplement. Both lysine and hydroxylysine inhibited the first enzyme of the pathway, aspartokinase. Unlike the Escherichia coli enzyme, S. faecalis dihydrodipicolinic acid synthetase was not inhibited by either lysine or hydroxylysine. Both amino acids caused the repression of dihydrodipicolinic acid synthetase and diaminopimelic acid decarboxylase. Failure of Lys(+)OHLys(s) strain to grow in hydroxylysine-supplemented medium was caused by the mimicking of lysine control by hydroxylysine. Because hydroxylysine could not completely substitute for lysine and lysine could not be synthesized, the organism did not grow. We tested three lysine analogues and found that they prevented lysine-depletion lysis in the Lsy(-)OHLys(s) strain, as did hydroxylysine. Each analogue seemed to support cell wall mucopeptide synthesis, although ornithine did not. Preliminary data indicated that these analogues like hydroxylysine, have growth-inhibitory action on the Lys(+)OHLys(s) strain, but not the Lys(+)OHLys(r) strain. The nature of the specificity of the lysine-adding enzyme for cell wall mucopeptide synthesis is discussed.     

Lysine production byBrevibacterium linens and its mutants: Activities and regulation of enzymes of the lysine biosynthetic pathway

Activity and regulation of key enzymes of the lysine biosynthetic pathway were investigated inBrevibacterium linens, a natural excretor of lysine, its lysine-overproducing homoserine auxotroph (Hom(-1)) and its auxotrophic and multianalogue-resistant high-yielding mutant (AEC NV 20(r)50). The activity of aspartate kinase (AK) and aspartaldehydate dehydrogenase (AD) was maximum during the mid-exponential phase of growth and decreased therafter. The mutants showed 10 and 20% more activity of AK and AD than the wild-type lysine excretor.B. linens (natural excretor) has a single AK and AD repressed and inhibited bivalently by lysine and threonine. Lysine slightly repressed and inhibited dihydrodipicolinate synthase (DS) and diaminopimelate decarboxylase (DD) of the wild type and of the mutant Hom(-1). The mutant AEC NV 20(r)50 showed DS and DD to be insensitive to lysine inhibition and repression. Persistence of a major part of the maximal activity of these enzymes during the late stationary phase of growth allowed prolonged synthesis and excretion of lysine. Stepwise addition of resistance to the different analogues of lysine in the mutant AEC NV20(r)50 resulted in an increase of enzyme activity and reduced repressibilities of enzymes that contributed to the high yield of lysine.     

Lysine overproduction mutations in the yeast Saccharomyces cerevisiae and its transfection into industrial Yeast strains ]

Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature. A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants. The intracellular content of lysine was also increased by 30%. The genetic nature of lysine overproduction was studied in this strain. An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1R) and the other is involved in the regulation of lysine production (PRL). Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome. Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses. The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.     

Mannan synthetase activity in three strains of Candida albicans

Abstract Mannan synthetase activity has been investigated in Candida albicans , strain 4918, as well as in two relatively avirulent, cerulenin-resistant mutant derivative strains, 4918-2 and 4918-10. In addition, investigations pertaining to the effects of the agents, cerulenin and sodium butyrate, on the level of mannan synthetase activity during the yeast to hyphal transition of these strains have been performed. The results show that mannan synthetase activity in yeast cells of both mutant strains is consistently higher than that observed in the parental strain. Similarly, the profile of enzyme activity exhibited by the mutant strains as morphogenesis proceeds differs from that of the wild-type. Sodium butyrate has no significant effect on enzyme activity in these strains, but the presence of cerulenin results in alterations in mannan synthethase activity during morphogenesis of strain 4918.     

Nitrate regulation of alpha-aminoadipate reductase formation and lysine inhibition of its activity in Penicillium chrysogenum and Acremonium chrysogenum

alpha-Aminoadipate reductase (alpha-AAR) is a key enzyme in the branched pathway for lysine and beta-lactam biosynthesis of filamentous fungi since it competes with alpha-aminoadipyl-cysteinyl-valine synthetase for their common substrate L-alpha-aminoadipic acid. The alpha-AAR activity in two penicillin-producing Penicillium chrysogenum strains and two cephalosporin-producing Acremonium chrysogenum strains has been studied. The alpha-AAR activity peaked during the growth-phase preceding the onset of antibiotic production, which coincides with a decrease in alpha-AAR activity, and was lower in high penicillin- or cephalosporin-producing strains. The alpha-AAR required NADPH for enzyme activity and could not use NADH as electron donor for reduction of the alpha-aminoadipate substrate. The alpha-AAR protein of P. chrysogenum was detected by Western blotting using anti-alpha-AAR antibodies. The mechanism of lysine feedback regulation in these two filamentous fungi involves inhibition of the alpha-AAR activity but not repression of its synthesis by lysine. This is different from the situation in yeasts where lysine feedback inhibits and represses alpha-AAR. Nitrate has a strong negative effect on alpha-AAR formation as shown by immunoblotting studies of alpha-AAR. The nitrate effect was reversed by lysine.