Thialysine and selenalysine utilization for protein synthesis by E. coli in comparison with lysine

Utilization of thialysine and selenalysine for protein synthesis by a lysine requiring E. coli mutant was studied. Incorporation into proteins of thialysine or selenalysine, added to culture medium together with lysine, becomes evident when the amount of available lysine in the medium is highly reduced, that is the mutant utilizes the isologs only after all the available natural aminoacid has been utilized. Compared to selenalysine, thialysine is better utilized; when both isologs are present in the medium at equal concentrations, up to 46% of protein lysine is substituted by thialysine and only 12% by selenalysine.     

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.     

Thialysine utilization by E. coli and its effects on cell growth

Summary Thialysine and selenalysine, two lysine isologs having the -methylene group substituted by a sulfur or a selenium atom, respectively, inhibit E. coli lysine-sensitive aspartokinase. The inhibition is specific, reversible and non-competitive. Compared to lysine, the two isologs have a less marked inhibitory effect, but show a similar homotropic cooperativity with a Hill's coefficient of about 2. The inhibition by each isolog is additive to that by lysine. Both compounds protect the enzyme against thermal inactivation. Overall, the data reported indicate that thialysine and selenalysine bind to the same allosteric site of lysine, the physiological modulator of the enzyme.     

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.     

Selenalysine and protein synthesis.

Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.     

Transport systems for lysine, thialysine and selenalysine in E. coli KL16

Two lysine transport systems have been identified in E. coli KL16. They differ in their affinity for lysine, one showing a KM of 0.36 microM and the other a KM of 4.7 microM. Different compounds with chemical similarities to lysine were tested for their capacity to interfere with lysine transport. Among these only thialysine and selenalysine competitively inhibit lysine transport. The inhibition is on both transport systems. Thialysine shows a KI of 4 microM for the low affinity system and a KI of 8 microM for the high affinity system. Selenalysine shows values of 6 microM and 12 microM respectively.     

Aspartokinase III repression in a thialysine-resistant mutant of E. coli

A thialysine-resistant mutant of the E. coli KL16 strain was isolated. It can grow equally well in the presence and in the absence of thialysine. The properties of the two lysine transport systems, of the lysyl-tRNA synthetase and of the aspartokinase III (AK III) were studied in the mutant and in the parent strain. AK III is the first enzyme of the lysine biosynthetic pathway and its activity is involved in the regulation of lysine biosynthesis by feed-back and repression mechanism. No difference between the two strains was evidenced as regards 1) the affinity of the transport systems for lysine and thialysine 2) the activity of the lysyl-tRNA synthetase 3) the allosteric inhibition of the AK III by lysine and thialysine. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the enzyme is much less repressed both by lysine and thialysine. The possible correlation between the activity of AK III and the thialysine-resistance is discussed in this paper.     

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.     

Intracellular transport of thialysine and selenalysine in CHO cells

The intracellular transport of thialysine and selenalysine in CHO cells has been studied. Data have been obtained indicating that the two lysine analogs can be transported by both the cationic aminoacid transport system and by the L transport system. The affinity of the cationic aminoacid transport system is similar for the two lysine analogs but lower than that for lysine and the affinity of the L transport system for the two lysine analogs is lower than that for leucine.     

Selenalysine utilization by CHO cells

CHO cells allowed to grow in a medium containing selenalysine can utilize it for protein synthesis. Selenalysine is incorporated into cell proteins in substitution of lysine: a maximum of 5% of protein lysine can be substituted. Protein lysine substitution by selenalysine can be correlated to the reduced viability of cells grown in its presence.     

Regulation of glucose transport in Candida utilis

The transport systems for glucose present in Candida utilis cells, growing in batch and continuous cultures on several carbon sources, have been studied. Two different systems were found: a proton symport and a facilitated diffusion system. The high-affinity symport (Km for glucose about 15 microM) transported one proton per mole of glucose and was partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol and glycerol. It was also induced by glucose concentrations up to 0.7 mM and repressed by higher ones. The level of repression depended on the external glucose concentration at which cells had grown in a way similar to that shown by the maltose-uptake system, so both systems seem to be under a common glucose control. Initial uptake by facilitated diffusion, the only transport system present in cells growing at glucose concentrations higher than 10 mM, showed a complex kinetic dependence on the extracellular glucose concentration. This could be explained either by the presence of at least two different systems simultaneously active, one with a Km around 2 mM and the other with a Km of about 1 M, or by the allosteric or hysteretic behaviour of a single carrier whose apparent Km would oscillate between 2 and 70 mM.     

Selenalysine utilization for growth and protein synthesis by a lysine requiring E. coli mutant

Summary Selenalysine can be utilized in substitution of lysine by a lysine requiring E. coli mutant. The presence of some lysine in the culture medium is necessary to allow selenalysine utilization for growth; in the presence of an excess of lysine, selenalysine is not utilized. When utilized, selenalysine gives rise to an increase of final growth. However, it shows some toxic effects as demonstrated by the decrease of both growth rate and cell viability. Selenalysine is incorporated into proteins in substitution of lysine. Up to a maximum of 50% of total protein lysine can be substituted. The decrease of cell viability is correlated with the extent of lysine substitution.This paper is dedicated to Professor A. E. Braunstein on his 80th birthday.     

Thialysine and selenalysine incorporation into CHO cell proteins and its effects on cell growth

CHO cells can incorporate into proteins both thialysine and selenalysine when both are present together in the culture medium. Thialysine and selenalysine inhibit cell growth and cell viability. The inhibitory effect of either analog is additive. The inhibition of cell viability is related to the extent of protein lysine substitution by thialysine or selenalysine; it is however irrelevant whether lysine is substituted by one or the other analog or by both.     

The regulation of transport of glucose, gluconate and 2-oxogluconate and of glucose catabolism in Pseudomonas aeruginosa.

1. The induction by glucose and gluconate of the transport systems and catabolic enzymes for glucose, gluconate and 2-oxogluconate was studied with Pseudomonas aeruginosa PAO1 growing in a chemostat under conditions of nitrogen limitation with citrate as the major carbon source. 2. In the presence of a residual concentration of 30mM-citrate an inflowing glucose concentration of 6-8 mM was required to induce the glucose-transport system and associated catabolic enzymes. When the glucose concentration was raised to 20mM the glucose-transport system was repressed, but the transport system for gluconate, and at higher glucose concentrations, that for 2-oxogluconate, were induced. No repression of the glucose-catabolizing enzymes occurred at the higher inflowing glucose concentrations. 3. In the presence of 30mM-citrate no marked threshold concentration was required for the induction of the gluconate-transport system by added gluconate. 4. In the presence of 30mM-citrate and various concentrations of added glucose and gluconate, the activity of the glucose-transport system accorded with the proposal that a major factor concerned in the repression of this system was the concentration of gluconate, produced extracellularly by glucose dehydrogenase. 5. This proposal was supported by chemostat experiments with mutants defective in glucose dehydrogenase. Such mutants showed no repression of the glucose-transport system by high inflowing concentrations, but with a mutant apparently defective only in glucose dehydrogenase, the addition of gluconate caused repression of the glucose-transport system. 6. Studies with the mutants showed that both glucose and gluconate can induce the enzymes of the Entner-Doudoroff system, whereas for the induction of the gluconate-transport system glucose must be converted into gluconate.     

Mutant of Escherichia coli K-12 Defective in the Transport of Basic Amino Acids

Escherichia coli K-12 possesses two active transport systems for arginine, two for ornithine, and two for lysine. In each case there is a low- and a high-affinity transport system. They have been characterized kinetically and by response to competitive inhibition by arginine, lysine, ornithine and other structurally related amino acids. Competitors inhibit the high-affinity systems of the three amino acids, whereas the low-affinity systems are not inhibited. On the basis of kinetic evidence and competition studies, it is concluded that there is a common high-affinity transport system for arginine, ornithine, and lysine, and three low-affinity specific ones. Repression studies have shown that arginine and ornithine repress each other's specific transport systems in addition to the repression of their own specific systems, whereas lysine represses its own specific transport system. The common transport system was found to be repressible only by lysine. A mutant was studied in which the uptake of arginine, ornithine, and lysine is reduced. The mutation was found to affect both the common and the specific transport systems.     

Regulation of lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis.

Further studies on the expression of the two aspartokinase activities in Bacillus bovis are presented. Aspartokinase I (previously shown to be inhibited and repressed by lysine) was found to be repressed by diaminopimelate in the wild-type strain. However, in a mutant unable to convert diaminopimelate to lysine, starvation for lysine resulted in an increase in aspartokinase I activity. Thus, lysine itself or an immediate metabolite was the true effector of repression. Aspartokinase II (previously shown to be inhibited by lysine plus threonine) was repressed by threonine. Studies with the parent strain and auxotrophs inidicated that only threonine or an immediate metabolite of threonine was involved in this repression. Methionine and isoleucine were not effectors of any of the detected aspartokinase activities. Apart from inhibition and repression controls, a third as yet undefined regulatory mechanism operated to decrease the levels of both aspartokinases as growth declined, even in mutants in which repression control was absent. In thiosine-resistant, lysine-excreting mutants with elevated levels of aspartokinase, the increase in activity could always be attributed to one enzyme or the other, never both. The existence of separate structural genes for each aspartokinase is therefore suggested.     

Degradation of thialysine- or selenalysine-containing abnormal proteins in CHO cells

CHO cells can incorporate thialysine and selenalysine in their proteins in substitution of lysine. Data are reported in the present paper showing that proteins containing either thialysine or selenalysine are unstable and quite rapidly degraded. The degradation rate is strictly related to the extent of protein lysine substitution. At similar extent of substitution, selenalysine-containing proteins are more unstable that thialysine-containing ones.     

Effect of exogenous cycllc amp on catabolite repression of cellulase formation in aspergillus nidulans

The addition of 1 mM cyclic AMP to induced and repressed cultures of Aspergillus nidulans and its mutant strain (CRR 141) resistant to catabolite repression was fully capable of releasing the wild type from catabolite repression while it caused hyperproduction of cellulases in glycerol repressed cultures. The relief of the catabolite repression was also accompanied by a dramatic drop in enhanced protease levels, thereby indicating that the synthesis of proteases (during the catabolite repression) is under the control of cyclic AMP.     

Utilization of lysine analogs by a lysine-requiring E. coli mutant

Thialysine and selenalysine cannot substitute lysine as a growth factor for a lysine-requiring E. coli mutant, but can nevertheless be utilized for protein synthesis in the presence of lysine. In order to have information about the effects of lysine on the utilization of the two analogs, the extent of the incorporation of the three aminoacids into newly synthesized proteins has been determined. The analog starts to be utilized by cells growing in a medium containing either analog and lysine when lysine concentration becomes very low. Of the two analogs, thialysine is more easily utilized. In fact thialysine can be utilized when the lysine/thialysine ratio in the medium is 1/25. Selenalysine starts to be utilized when the lysine/selenalysine ratio is 1/200.     

Amino acid transport during development of Neurospora crassa conidia: Substrate repression

The increasing amino acid transport activity which occurs during germination of Neurospora crassa is repressed by substrate amino acid. This repression acts on the transport systems similarly to competition in that amino acids within a specific transport class (e.g., basic) repress that system. Repression of the other system (neutral-aromatic) by that amino acid is shown to be repression of the general transport system. The level of repression and the rate of derepression after removal of the amino acid appear to depend on the nonrepressed level and rate. The extent of repression caused by increasing the concentration of the amino acid is shown to be different for two amino acids. A mutant deficient in developmental transport for arginine and phenylalanine contains two mutations. The mutation affecting phenylalanine transport maps on linkage group III and results in an accumulation of phenylalanine in the medium, thus repressing the development of this transport activity.This work was supported in part by a National Institutes of Health, U.S. Public Health Service Traineeship in Genetics (2-T01-GM1316).