Overproduction and purification of lysyl-tRNA synthetase encoded by the herC gene of E coli.

Lysyl-tRNA synthetases are synthesized from two distinct genes in E coli, lysS and lysU, but neither gene product has been purified distinctively by using overproducing systems. The lysS gene has been identified by a herC mutation which restores maintenance of the mutant ColE1 replicon. The herC gene product was overproduced by using a tac promoter fusion and purified to homogeneity. The purified HerC protein possesses a lysyl-tRNA synthetase activity as predicted by the sequence identity of herC to lysS. The procedure is useful for rapid mass-scale purification of lysyl-tRNA synthetase.     

Roles of the two lysyl-tRNA synthetases of Escherichia coli: analysis of nucleotide sequences and mutant behavior.

The complete nucleotide sequence of lysU, the gene for the heat-inducible lysyl-tRNA synthetase of Escherichia coli, was determined and compared with the published sequence of lysS (herC), the gene for the constitutive lysyl-tRNA synthetase. These unlinked genes were found to be identical over 72% of their lengths. The deduced amino acid sequences of the respective gene products, LysU and LysS, were identical over 85% and similar over 92% of their lengths. Accumulation of high levels of LysU during growth of strains carrying the wild-type allele of lysU on multicopy plasmids had no observable effect on growth or on the synthesis of LysS. A lysU deletion strain was constructed and was shown to grow normally at low temperature (28 degrees C) but poorly at 44 degrees C; the slow growth (45% of normal) at elevated temperature was fully reversed by plasmids bearing wild-type lysU. The implications of these findings for the existence of two aminoacyl-tRNA synthetases for lysine are discussed.     

Characterization of a Mycoplasma hominis gene encoding lysyl-tRNA synthetase (LysRS)

Abstract The gene encoding lysyl-tRNA synthetase ( lysS ) in Mycoplasma hominis was cloned and sequenced. The gene was found to have an open reading frame of 1466 bp encoding a polypeptide with a predicted molecular mass of 57 kDa. The amino acid sequence showed 44.3% and 43.7% identity to the Escherichia coli lysyl-tRNA synthetases, encoded by lysS and lysU . Only one lysyl-tRNA synthetase encoding gene was found in M. hominis . The G+C content of the gene was found to be 28.6%, which is significantly lower than in other prokaryotes. The gene was located 4 kb upstream of the M. hominis PG21 rRNA B operon.     

Homology of lysS and lysU, the two Escherichia coli genes encoding distinct lysyl-tRNA synthetase species.

In Escherichia coli, two distinct lysyl-tRNA synthetase species are encoded by two genes: the constitutive lysS gene and the thermoinducible lysU gene. These two genes have been isolated and sequenced. Their nucleotide and deduced amino acid sequences show 79% and 88% identity, respectively. Codon usage analysis indicates the lysS product being more efficiently translated than the lysU one. In addition, the lysS sequence exactly coincides with the sequence of herC, a gene which is part of the prfB-herC operon. In contrast to the recent proposal of Gampel and Tzagoloff (1989, Proc. Natl. Acad. Sci. USA 86, 6023-6027), the lysU sequence is distinct from the open reading frame located adjacent to frdA, although large homologies are shared by these two genes.     

The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes.

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS+ lysU+) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS+ delta lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginose and B, megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.     

Partial characterization of a lysU mutant of Escherichia coli K-12.

The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity. Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent. When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed. The lysU mutation was transferred to other E. coli K-12 backgrounds by using P1 transduction. The lysU transductants behaved comparably to their lysU+ parents at different growth temperatures. Therefore, the LysU proteins does not appear to be essential for growth at high temperatures, at least under the conditions examined here. In addition, lysU transductants were found to be defective for inducible lysine decarboxylase, (LDC), inducible arginine decarboxylase (ADI), and melibiose utilization (Mel), which are all missing in GNB10181. Complementation of the above missing functions was achieved by using the Clarke-Carbon plasmids pLC4-5 (LysU LDC) and pLC17-38 (LysU Mel ADI). From these experiments, it appears that GNB10181 has suffered a chromosomal deletion between 93.4 and 93.7 min, which includes the lysU gene. By using plasmid pLC17-38, the position of ADI on two-dimensional gels was identified. Finally, lysS delta lysU double mutants were constructed which can potentially be used as positive selection agents for the isolation of LysRS genes from other sources.     

Thermal death of temperature-sensitive lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants of Bacillus subtilis: effect of culture medium and developmental stage

The growth of thermosensitive Bacillus subtilis lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants (lysS1 and trypS1) (l-lysine:transfer ribonucleic acid [tRNA] ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) was terminated when exponential phase cells were shifted from 30 to 43 C in a rich medium. Under these conditions, the temperature-inhibited cells undergo thermal death; they rapidly lose their ability to form colonies at 30 C. Another lysyl-tRNA synthetase mutant (lysS2) is refractory to thermal death even though its growth is inhibited at 43 C. The thermal death response of the lysS1 mutant is affected by the stage of cell development. At periods in spore outgrowth and sporogenesis these cells become refractory to thermal death. The refractory state does not result from the production of an inhibitor, or from the degradation of an activator of thermal death. However, culture medium composition does modify the thermal death response. Rich media enhance the effect, and no thermal death occurs in the lysS1 strain grown in a minimal medium. Temperature-sensitive cells can grow in a lysine- (0.25 mM) or tryptophan- (0.25 mM) supplemented minimal medium at 43 C, but amino acid concentrations of 25 mM only transiently protect trypS1 and lysS1 cells from thermal death in a rich medium. Osmotic agents such as sucrose (0.5 M) and NaCl (0.34 M) completely prevent thermal death in the lysS1 strain, although growth is still arrested. On solid media, sucrose stabilized lysS1 cells can form colonies at the restrictive temperature, but neither sucrose (0.5 M) nor NaCl (0.34 M) stabilized the lysS1 enzyme in vitro. Chloramiphenicol increased the rate of thermal death of the lysS1 strain but decreased the thermal death response of the trypS1 mutant. Considering the nature of the enzyme defect in the lysS1 strain, the common genetic origin of the spore and vegetative lysyl-tRNA synthetase, and the protective effects exerted by lysine and osmotic agents, it is tentatively concluded that thermal death results from irreversible inactivation of the mutant gene product. According to this hypothesis, either the lysS1 enzyme is altered during sporogenesis or some physiological or structural aspect of this developmental phase can stabilize the mutant phenotype and thereby rescue cells from thermal death.     

Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product.

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.     

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.     

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.     

The lrp gene product regulates expression of lysU in Escherichia coli K-12.

In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant. Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent. The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion. A region extending over 100 nucleotides, between 60 and 160 nucleotides upstream from the start of the lysU coding sequence, showed altered sensitivity to DNase I digestion in the presence of the Lrp protein. The extent of protected DNA suggests a complex interaction of Lrp protein and upstream lysU DNA.     

Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12.

The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.     

Abnormal induction of heat shock proteins in an Escherichia coli mutant deficient in adenosylmethionine synthetase activity.

Most prototrophic strains of Escherichia coli become restricted for methionine at 44 degrees C. A mutant strain (RG62 metK) in which the level of S-adenosylmethionine synthetase activity is only 10 to 20% of normal shows constitutive expression of one of the heat shock proteins, the lysU gene product, lysyl-tRNA synthetase form II, at 37 degrees C. These findings suggested a possible linkage between methionine metabolism and heat shock. We examined the induction of heat shock polypeptides in strain RG62 (metK) and in its parent, RG (metK+), from which it was derived by spontaneous mutation. Exponential-phase cultures of the two strains were pulse-labeled with [3H]leucine shortly after a shift from 37 to 44 degrees C, and the total cellular polypeptides were examined by two-dimensional electrophoresis. The results confirmed the constitutive production of the lysU gene product previously reported for strain RG62, but also revealed that the induction of 2 of the 17 heat shock polypeptides, C14.7 and G13.5, was markedly depressed. Otherwise the heat shock induction pattern was similar in timing and magnitude in the two strains. Transformation of the mutant strain with a plasmid, pK8, containing the metK coding sequence and promoter region as a 1.8-kilobase insert into pBR322 restored normal induction of C14.7 and G13.5, but did not prevent constitutive expression of the lysU gene product in the medium required for growth of this strain. The three heat shock polypeptides abnormally controlled in strain RG62 are the three polypeptides which are not induced when rapid synthesis of the htpR gene product is induced by isopropyl-beta-D-thiogalactopyranoside at 28 degree C (R. A. VanBogelen, M. A. Acton, and F. C. Neidhardt, Genes Dev. 1:525-531, 1987). We postulate that induction of these three polypeptides involves metabolic signals in addition to the synthesis of the htpR gene product and that strain RG62 (metK) fails to produce the signals involved in induction of C14.7 and G13.5 on a shift-up in temperature and produces the signal related to lysU induction even at 37 degree C.     

Gene for Heat-Inducible Lysyl-tRNA Synthetase (lysU) Maps near cadA in Escherichia coli

A hybrid ColE1 plasmid from the Clarke-Carbon colony bank with a 7-kilobase insertion was found to encode the inducible lysyl-tRNA synthetase along with the catabolic enzyme lysine decarboxylase. The gene for the inducible synthetase, lysU, must lie within 0.3 min of the lysine decarboxylase gene, cadA, at 92 min on the Escherichia coli genetic map.     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.     

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 location of two mutations affecting the lysyl-transfer ribonucleic acid synthetase of Bacillus subtilis

Two mutations (lysS1 and lysS2), each independently resulting in a thermosensitive, lysyl-transfer RNA synthetase (l-lysine: tRNA ligase [adenosine 5'-monophosphate] EC 6.1.1.6), have been mapped on the Bacillus subtilis chromosome between purA16 (adenine requirement) and sul (sulfanilamide resistance). They are linked by transformation with sul (70 to 74% cotransfer) in the order purA16-lysS1-lysS2-sul. The mutant loci are either in the same gene or in two closely linked genes. They are not linked to the tryptophanyl-tRNA synthetase structural gene or to the lys-1 locus.