©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Comparison of the Enzymatic Properties of the Two Escherichia coli Lysyl-tRNA Synthetase Species (*)

Annie Brevet , Josiane Chen , Franoise Lévque , Sylvain Blanquet , Pierre Plateau (§)

From the (1)Laboratoire de Biochimie, URA 240 CNRS, Ecole Polytechnique, 91128 Palaiseau Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In Escherichia coli, lysyl-tRNA synthetase activity is encoded by either a constitutive lysS gene or an inducible one, lysU. The two corresponding enzymes could be purified at homogeneity from a lysU and a lysS strain, respectively. Comparison of the pure enzymes, LysS and LysU, indicates that, in the presence of saturating substrates, LysS is about twice more active than LysU in the ATP-PP exchange as well as in the tRNA aminoacylation reaction. Moreover, the dissociation constant of the LysU-lysine complex is 8-fold smaller than that of the LysS-lysine complex. In agreement with this difference, the activity of LysU is less sensitive than that of LysS to the addition of cadaverine, a decarboxylation product of lysine and a competitive inhibitor of lysine binding to its synthetase. This observation points to a possible useful role of LysU, under physiological conditions causing cadaverine accumulation in the bacterium. Remarkably, these conditions also induce lysU expression.

Homogeneous LysU and LysS were also compared in ApA synthesis. LysU is only 2-fold more active than LysS in the production of this dinucleotide. This makes unlikely that the heat-inducible LysU species could be preferentially involved in the accumulation of ApA inside stressed Escherichia coli cells. This conclusion could be strengthened by determining the concentrations of ApN (N = A, C, G, or U) in a lysU as well as in a lysU strain, before and after a 1-h temperature shift at 48 °C. The measured concentration values were the same in both strains.


INTRODUCTION

In a cell, the covalent attachment of one amino acid to its cognate tRNAs is, in principle, performed by a single aminoacyl-tRNA synthetase. A rare exception to this rule is the case of Escherichia coli lysyl-tRNA synthetase (LysRS),()which occurs as two distinct species(1, 2) . The corresponding genes, lysS and lysU, are submitted to different regulations.

lysU expression is induced (i) by the addition of alanine, leucine, or leucine-containing dipeptides in the growth medium(3, 4, 5) or (ii) by exposure to either anaerobiosis(6) , high temperature(7) , or low external pH (8, 9) conditions. Recently, lysU expression was shown to be under the negative control of the leucine-responsive regulatory protein (Lrp)(10, 11, 12) , a global regulatory factor that would participate to the adaptation of E. coli to its environment in the host intestinal tract(13) . The effect of this regulatory protein on lysU expression is antagonized by the presence of leucine(14) .

The lysS gene makes part of a dicistronic transcriptional unit comprising also prfB(15) . The latter gene encodes the peptide chain release factor specific of stop codons UAA and UGA. The translation of prfB is tightly autoregulated by a mechanism requiring a +1 frameshift at a UGA codon(16) . Because of the proximity between the stop codon of the prfB open reading frame and the downstream initiator codon of lysS(15) , the expression of lysS is likely to be influenced by the translation of prfB. Besides, lysS does not respond to the above physiological conditions which induce lysU expression(1, 17) .

Gene disruption experiments showed that each of the lysS and lysU genes is dispensable for the growth of the bacterium. lysU cells do not display a clear phenotype. Clark and Neidhardt reported that the growth of a lysU strain at 44 °C is slightly slower than that of the isogenic lysU strain(18) . However, this difference could not be reproduced in other genetic contexts(6, 19) . Disruption of lysS renders the cell cold-sensitive(6, 20) . This behavior can be explained by the low expression of lysU at growth temperatures below 37 °C. Accordingly, transformation of a lysS strain with a plasmid overexpressing lysU is enough to cure the cold-sensitive phenotype(6) .

The sequences of the two E. coli LysRS species share 88.5% identity, with 447 identical amino acids out of a total of 505 (Ref. 2 and corrected sequence of LysU under EMBL/GenBank accession number X16542). The homology is, however, smaller than in the case of other E. coli isoenzymes. For instance, the two EF-Tu species or the two glutamate decarboxylases display 99.7 and 98.9% identity, respectively(21, 22) . Therefore, the question arises to know whether the two E. coli LysRSs may be functionally distinct. One possibility is that the lysU product has features directly or indirectly useful for the cell to adapt to extreme conditions such as high temperatures, high pH, or anaerobiosis.

In the present study, advantage was taken of the availability of lysS and lysU strains to prepare samples of either the LysU or the LysS protein and to compare their kinetic properties. The most noticeable difference is an affinity of LysU for lysine nearly 8-fold higher than that of the LysS species. In agreement with this difference, LysU activity is relatively less sensitive than the LysS one to the presence of cadaverine, an inhibitor of lysine binding. The latter polyamine is produced at the expense of lysine in several stressing cellular conditions. Remarkably, most of these conditions induce lysU expression too.


EXPERIMENTAL PROCEDURES

Materials

L-[C]Lysine (11.1 GBq/mmol) was from Amersham, [P]PP (300 GBq/mmol) was from DuPont NEN. ApA was from Boehringer, cadaverine (1,5-diaminopentane) was from Aldrich. Unfractionated E. coli tRNA was from Boehringer, and purified E. coli tRNA (1000 pmol of lysine acceptance/A unit) from Sigma. Hydroxylapatite was from Bio-Rad and DEAE-Sephadex A50 from Pharmacia.

Polyacrylamide gel electrophoresis analyses were performed with the Pharmacia Phast gel apparatus under the conditions recommended by the supplier.

Bacterial Strains

The bacterial strains used in this study included XA103 (F (lac-pro) gyrA rpoB metB argE(Am) ara supF)(23) , PAL2103UKTR (F (lac-pro) gyrA rpoB metB argE(Am) ara supF lysU::kan srl-300::Tn10 recA56) and PAL3103SKTR (F (lac-pro) gyrA rpoB metB argE(Am) ara supF lysS::kan srl-300::Tn10 recA56)(6) . For the purifications of LysS and LysU, strain PAL2103UKTR transformed by plasmid pXLysKS1 (lysS) (2) and strain PAL3103SKTR transformed by pXLys5 (lysU) (24) were used, respectively.

Purification of LysS and LysU from Overproducing Strains

All steps were performed at 4 °C in buffers systematically containing 10 mM 2-mercaptoethanol and 0.1 mM EDTA. The cells from an overnight culture were harvested by centrifugation during 45 min at 5000 g. The pellet was suspended in 20 mM Tris-HCl buffer (pH 7.8) at a cell density of 0.15 g (wet weight)/ml of buffer and sonicated for 10 min. Cell debris were removed by centrifugation at 10,000 g for 20 min. Then, streptomycin sulfate was added to the supernatant at a final concentration of 27 mg/ml. After centrifugation of the sample for 20 min at 10,000 g, the supernatant was brought to 80% ammonium sulfate saturation and centrifuged for 30 min at 10,000 g. The protein pellets were dissolved in 20 mM potassium phosphate buffer (pH 6.75) (buffer A) and dialyzed against the same buffer.

Hydroxylapatite chromatography of the dialyzed proteins was performed through a column of 2.6 19 cm equilibrated in buffer A. After a 300-ml wash with buffer A, elution was carried out at a flow rate of 30 ml/h using a linear gradient of potassium phosphate concentration (2.5 liters, from 20 to 300 mM). Fractions displaying LysRS activity were pooled and dialyzed against buffer A.

At this stage, the LysS protein was already more than 95% pure, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In the case of LysU, further chromatographic steps were required; after ammonium sulfate precipitation (80%) and subsequent dialysis against buffer A, the protein sample containing LysU (20 ml) was applied onto a DEAE-Sephadex column (1.3 3.8 cm; 4.2 ml/h) equilibrated in 50 mM potassium phosphate (pH 6.75) (buffer B). The column was washed with 5 ml of buffer B containing 100 mM KCl. Elution was performed with a linear gradient of KCl concentration in buffer B (125 ml, from 100 to 500 mM KCl). Fractions exhibiting LysU activity were pooled (20 ml) and directly loaded onto a hydroxylapatite column (2 4.8 cm; 6 ml/h) equilibrated in buffer A. The column was washed with buffer A, and LysU was eluted by a linear gradient of potassium phosphate (600 ml, from 50 to 300 mM). Active fractions were pooled (100 ml) and dialyzed against buffer A.

For storage at -20 °C, the enzymes (10 mg/ml) were dialyzed against 20 mM Tris-HCl buffer (pH 7.8) containing 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and 60% glycerol.

Characterization of the LysRS Subspecies in Various Strains

To analyze the LysRS subspecies produced by the lysS and lysU strains, crude extract preparation from 250-ml cultures, precipitation of nucleic acids with streptomycin sulfate and precipitation of proteins with ammonium sulfate were performed as described above. A hydroxylapatite chromatography was further performed on a 1.7 4-cm column equilibrated in buffer A. After a 50-ml wash with buffer A, elution was insured at a flow rate of 10 ml/h using a linear gradient of potassium phosphate concentration (225 ml, from 20 to 300 mM).

In the case of the lysU extracts, the hydroxylapatite chromatography produced three peaks of LysRS activity each of which was dialyzed against 20 mM potassium phosphate buffer (pH 8.2) and applied onto a DEAE-Sephadex A50 column (0.3 9 cm) equilibrated in 20 mM potassium phosphate buffer (pH 8.2). Elution was performed at a flow rate of 0.5 ml/h using a linear gradient of potassium phosphate concentration (10 ml, from 20 to 500 mM).

Enzymatic Assays

Unless otherwise stated, initial rates of tRNA aminoacylation were measured at 37 °C in 100 µl of 20 mM Tris-HCl buffer (pH 7.8), 150 mM KCl, 2 mM ATP, 0.1 mM EDTA, 7 mM MgCl, 5 kBq L-[C]lysine, 30 µM lysine, and 150 µM unfractionated E. coli tRNA. After incubation in the presence of catalytic amounts of enzyme, tRNA was precipitated with trichloroacetic acid, filtered, and counted for incorporated C as described previously(25) . One unit of LysRS activity is the amount of enzyme producing 1 µmol of lysyl-tRNA/min.

Enzyme specific activity was calculated using protein concentrations determined by the Bio-Rad protein assay, with bovine serum albumin as the standard. In the cases of pure LysS or LysU, concentrations were measured using a UV absorption coefficient of 0.5 A unitsmgml(24) .

Initial rates of the ATP-PP exchange reaction were measured at 25 °C in 100 µl of 20 mM Tris-HCl buffer (pH 7.8), and 0.1 mM EDTA, containing 3-15 kBq [P]PP plus various concentrations of ATP, lysine, and unlabeled PP. MgCl in the assay systematically exceeded by 3 mM the sum of the ATP and PP concentrations. The reaction was initiated by the addition of catalytic amounts of the LysRS under study. Labeled ATP was adsorbed on charcoal, filtered, and counted as described previously(26) .

The synthesis of ApA was assayed at 37 °C by bioluminescence(24) . The incubation mixture included 20 mM Tris-HCl buffer (pH 7.8), 150 mM KCl, 7 mM MgCl, 5 mM ATP, 100 µM lysine, 150 µM ZnCl, 0.04 mg/ml yeast pyrophosphatase (from Boehringer) and catalytic amounts of the LysRS under study.

All Michaelian parameters were derived from iterative nonlinear fits of the theoretical rate equations to the experimental values, using the Levenberg-Marquardt algorithm(27) .


RESULTS

Purification of LysS and LysU

To avoid any cross-contamination between LysS and LysU proteins, LysS was purified from a lysU null mutant overexpressing lysS, whereas LysU was purified from a lysS null mutant overexpressing lysU. Strains were grown overnight at 37 °C in 2 liters of 2 TY medium (28) containing 100 µg/ml ampicillin. In these conditions, the two above strains overproduced LysRS activity 200- and 36-fold, respectively, as compared with the parental strain XA103 (lysSlysU)(23) .

To purify LysS or LysU, a chromatographic step on hydroxylapatite was used. At this stage, a broad and asymmetrical peak of LysS activity suggested several subspecies of the enzyme (Fig. 1). Such a behavior was reminiscent of previous observations that E. coli LysRS eluted as multiple peaks on hydroxylapatite(29, 30, 31, 32) . To verify that the properties of the overproduced enzyme were identical to those of LysS of chromosomal origin, a control experiment was performed with the lysU strain PAL2103UKTR. With this strain, three distinct peaks of LysRS activity were observed (Fig. 1), which were separately pooled and further purified on DEAE-Sephadex columns. The three enzymes subspecies were indistinguishable according to their K values for lysine (at ATP and PP concentrations of 2 mM and 0.15 mM, respectively) and for ATP (2 mM lysine and 0.15 mM PP) in the ATP-PP exchange reaction. Consequently, a single pool was made from the broad asymmetrical peak obtained with the overproduced LysS enzyme. The pooled protein behaved homogeneous according to SDS-polyacrylamide gel electrophoresis analysis and showed K values for lysine and ATP identical to those measured with the three subspecies of LysS of chromosomal origin.


Figure 1: Hydroxylapatite chromatography of extracts of strain PAL2103UKTR (lysU) (A) and of the same strain transformed by plasmid pXLysKS1 harboring lysS (B). Columns were eluted using a linear gradient of potassium phosphate concentration from 20 to 300 mM (pH 6.75). The A of the column effluent was monitored (). Prior to tRNA aminoacylation measurements the fractions of the profiles A and B were diluted 80- and 20,000-fold, respectively ().



Overproduced LysU eluted from the hydroxylapatite column at 100 mM phosphate, a concentration significantly lower than that required to elute LysS (140 mM). The activity profile was symmetrical. A control experiment with a lysS strain showed a single LysU species of chromosomal origin also eluting at 100 mM phosphate (data not shown). To obtain homogeneous enzyme, the sample of overproduced LysU had to be further purified on DEAE-Sephadex and once more on hydroxylapatite.

These purification procedures yielded 140 and 25 mg of LysS and LysU, respectively, from 9 g of wet cells ().

ATP-PPExchange Reaction

Initial rates of LysS and LysU in the isotopic ATP-[P]PP exchange reaction were compared as a function of lysine, ATP, and PP concentrations. In a first set of experiments, apparent K values for lysine (K) were measured at various nonsaturating ATP concentrations, while PP concentration was fixed equal to 150 µM. Whatever the LysRS species and the ATP concentration studied, Michaelian kinetics were always observed. The apparent k and K values depended on the ATP concentration so that double-reciprocal plots produced lines which intersected on the left of the ordinate axis. The results with LysS are shown in Fig. 2. When PP concentration was varied in the presence of fixed concentrations of lysine (2 mM) and ATP (2 mM), Michaelian kinetics were obtained again with each enzyme, yielding K


Figure 2: Double-reciprocal plot of the initial rate of the ATP-PP exchange catalyzed by LysS enzyme versus lysine concentration in the assay. The initial rates were measured at ATP concentrations of 0.02 mM (), 0.1 mM (), 0.5 mM (), 1 mM (), or 2 mM (). The straight lines were calculated with parameters obtained through the fitting of the entire set of data to the reaction scheme. For the sake of clarity, only shown is the portion of the plot corresponding to the highest lysine concentrations and to the highest initial rates.



According to the following reaction scheme ()(26) ,

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

the experimental data could be fitted to the following relationship,

On-line formulae not verified for accuracy

where v is the initial rate of exchange, [E] is the total enzyme concentration, k is the catalytic constant of the reaction, and K = [E][Lys]/[E:Lys], C = [E:Lys][ATP]/[X], C = [E:LysAMP][PP]/[X], K = [E][ATP]/[E:ATP], C = [E:ATP][Lys]/[X] = CK/K.

Finite values of K and K could be deduced (), thereby suggesting random rather than ordered binding of ATP and lysine to the enzyme. A mechanism where lysine binds first would have produced an infinite value of K, and the lines in Fig. 2would have intersected on the ordinate axis. Similarly, a mechanism where ATP binds first would have produced an infinite value of K. However, a mechanism where one out of the two substrates would form an abortive complex with the enzyme cannot be ruled out since it would also be compatible with the above rate law (33).

The measured k and K values of LysU were respectively 2.3- and 1.6-fold lower than those of LysS. In contrast, the dissociation constant of lysine complexed to LysU in the absence of ATP (K) was nearly 8-fold smaller than that of the lysine-LysS complex. The value of C, which corresponds to the K value for lysine at saturation of both ATP and PP, was also smaller by a factor of 4 in the case of LysU as compared with its value in the case of LysS ().

Aminoacylation of tRNA

In another set of experiments, initial rates of tRNA aminoacylation by either LysS or LysU were compared. A Michaelian behavior was observed in the case of each LysRS. The K value of LysU for lysine (0.7 µM), measured at a fixed unfractionated tRNA concentration of 150 µM, was significantly lower than the K value of LysS (4.5 µM). At 30 µM lysine, the K value for purified tRNA was 2.6-fold higher with LysU than with LysS ().

ApA Synthesis

ApA is a representative member of a family of dinucleoside polyphosphates (ApN, where N = A, C, G, or U and n = 3 or 4) detectable in most if not all living cells (34) and known to be produced in vitro as well as in vivo by various aminoacyl-tRNA synthetases, including the LysRSs(35) . Because dinucleoside polyphosphates strongly accumulate in response to various stress conditions including heat shock(36) , we searched for whether the heat-inducible LysU protein could be more efficient than LysS in promoting ApA synthesis.

LysU sustained a rate of ApA synthesis only 2.1-fold higher than that with LysS (). Therefore, it is unlikely that the LysU species is preferentially involved in the ApN accumulation observed during a heat shock response. To reinforce this conclusion, the ApN contents of strains PAL2103UKTR (lysU) and XA103 (lysU) were compared before and after a temperature shift. Bacteria were grown aerobically in LB medium, at 37 °C. When the optical density of the culture reached 0.3 at 650 nm, ApN were extracted from an aliquot of the cultures and assayed. The ApN concentration amounted to 2.8 and 3 µM in the lysU and lysU strains, respectively. The remaining part of the culture was shifted from 37 to 48 °C. After 60 min at 48 °C, the ApN concentration in the lysU strain (11 µM) remained similar to that in the lysU strain (11 µM).

Inhibition by Cadaverine

In principle, a LysU protein having a higher affinity for lysine than that of LysS might be beneficial to the cell when intracellular lysine concentration becomes limiting. Alternatively, LysU might be useful to resist inhibition by a lysine analog. One such naturally occurring analog is cadaverine. Therefore, the initial rate of the ATP-PP exchange was measured at fixed concentrations of ATP (2 mM) and PP (2 mM), in the presence of various concentrations of lysine and cadaverine. In the case of both LysRSs, cadaverine behaved as a competitive inhibitor of lysine. The corresponding K values toward LysS and LysU were similar (53 and 73 µM, respectively). Consequently, at a saturating concentration of lysine, LysU was less sensitive to the inhibition by cadaverine than LysS, because of the lower K value for lysine of the former enzyme. Thus, at lysine concentrations greater than 7 µM, 50% inhibition of LysU activity required a cadaverine concentration at least six times greater than that causing 50% inhibition of LysS activity.

Thermostability

Because lysU gene expression accompanies the adaptation of the bacterium to thermal transition, it was of interest to compare the thermostabilities of LysU and LysS. This measurement was made in a 20 mM Tris-HCl buffer (pH 7.8) containing 7 mM MgCl, 0.1 mM EDTA, 2 mM ATP, 30 µML-lysine, and 150 µM of unfractionated E. coli tRNA. After incubation at 42 °C for various times, aliquots (100 µl) were removed from the assay and the initial velocity of tRNA aminoacylation was determined in a standard aminoacylation mixture. The kinetics of inactivation at 42 °C followed exponential curves (Fig. 3) with half-life times of LysS and LysU activities of 62 and 141 s, respectively, in agreement with previous results indicating that LysU was more resistant to thermal inactivation than LysS(32, 37) .


Figure 3: Comparison of the thermostabilities of LysS () and LysU (). Residual tRNA aminoacylation activity (expressed as percentage of the activity at time 0) is plotted as a function of the time of incubation. Enzymes (0.2 µM) were incubated at 42 °C in the presence of 20 mM Tris-HCl (pH 7.8), 0.1 mM EDTA, 7 mM MgCl, 2 mM ATP, 30 µML-lysine, and 150 µM unfractionated E. coli tRNA.




DISCUSSION

The reason for the occurrence of two LysRS genes in E. coli has to be searched for at the regulatory or the functional level. A first possibility is that the juxtaposition of two genes results in a finer regulation of cellular LysRS concentration. For instance, the coordination of lysS and prfB expressions might become harmful in some cases and the switch-on of a second LysRS gene would offer the possibility to escape the coupling between lysS and prfB. Alternatively, the addition of two LysRS genes can provide a functional advantage, provided the characteristics of each synthetase are adapted to different compositions of the cytoplasm. In such a case, however, some discrepancy should be recognizable when comparing the biochemical properties of LysS and LysU.

This study indicates that the greatest difference between LysS and LysU is at the level of their affinities for lysine, the dissociation constant of the LysU-lysine complex being 8-fold smaller than that of the LysS-lysine complex. In contrast, the binding parameters of ATP and purified tRNA are similar with the two synthetases. Although only one tRNA isoacceptor has yet been described(38) , in agreement with the sequence identity of the three E. coli tRNA genes(39) , it cannot be excluded that, under particular growth conditions, post-transcriptional modifications may render tRNA able to distinguish between LysS and LysU. For instance, the tRNA molecule has been shown to incorporate selenium when the growth medium contains micromolar amounts of this element(40) .

To evaluate the advantage of having two LysRS species with distinct K values for lysine, it is interesting to involve cadaverine, a polyamine produced from lysine by a decarboxylase. The cadA gene encoding lysine decarboxylase forms an operon with cadB, a gene corresponding to an antiporter protein capable of importing one lysine molecule while it excretes one cadaverine molecule(41) . The coupled actions of the cadA and cadB products is believed to be involved in the adaptation of the bacterium to low external pH, since the conversion of each lysine molecule to cadaverine consumes one proton(41) . However, this mechanism may transiently both decrease intracellular lysine and increase internal cadaverine. Since cadaverine behaves as a competitive inhibitor of lysine binding to LysRS (see ``Results''), a stimulated production of LysU, the activity of which is less sensitive to cadaverine than that of the constitutive LysS, could be useful.

To our knowledge, little information is available on the cadaverine concentration in E. coli cells. Igarashi et al. measured a cadaverine amount of 130 nmol/g of wet cells in the case of E. coli B cells grown in minimal medium without lysine(42) . With a mutant strain lacking the biosynthetic arginine decarboxylase (43) grown in another minimal medium without lysine, Kashiwagi and Igarashi found 2.3 nmol of cadaverine/mg of protein. These values, which correspond to 200-700 µM cellular cadaverine, are likely to increase under conditions capable of inducing lysine decarboxylase activity. Under appropriate conditions, the activity of this enzyme increases in the cell by a factor of up to 3000 (44), and lysine decarboxylase may represent nearly 2% of the total E. coli proteins(45) . Consequently, even if the major part of the produced cadaverine were excreted (or sequestered by binding to membrane phospholipids or nucleic acids), it would seem reasonable to imagine that, upon lysine decarboxylase induction, free cellular cadaverine concentration might reach values exceeding the K value of cadaverine toward any LysRS species (50 µM).

Interestingly, the regulation of lysU shares common features with that of the cadBA operon: (i) lysU and cadBA are induced by anaerobiosis or by low external pH(6, 8, 46) ; (ii) the induction of these genes occurs only when bacteria are grown in rich medium(6, 44) ; (iii) the expressions of both lysU and cadA genes are enhanced in mutants of hns(47, 48) and in mutants of cadR(18, 44) , a gene suspected to be identical to the lysP gene encoding lysine permease(49) . Moreover, (iv) homologous DNA sequences are found upstream of cadB and lysU(6) in regions shown to be important for the regulations of these genes(11, 50) . In turn, cadBA expression is induced by the addition of external lysine while lysU expression is not(45, 51) . The latter behavior is in agreement with the idea that, in the presence of a high supply of lysine, the derepression of lysU would become unnecessary. Although cadaverine will accumulate following cadBA induction, a high cellular lysine concentration would protect LysRS activity against inhibition by the polyamine.

If the two LysRS species of E. coli functionally account for various physiological conditions, the question arises why two genes have been selected during evolution to produce two synthetases, rather than post-translational modifications of a single gene product. In truth, evolution may have favored also the latter mechanism to multiply the diversity of LysRS species in E. coli. As shown here and already often noticed before, LysRSs appear as multiple subspecies upon hydroxylapatite chromatography (29-32). Two-dimensional gel electrophoresis experiments also revealed that the products of lysS and even of lysU may occur under several forms in vivo(1) . The biological significance of such variants, and the nature of the corresponding maturations remain mysterious. However, it reminds us the behavior of tryptophanyl-tRNA synthetase, which is also recovered as two distinct subspecies from hydroxylapatite chromatography(52) , and those of threonyl- and glutaminyl-tRNA synthetases, which are partly phosphorylated in vivo through a mechanism involving heat-shock proteins DnaJ and DnaK(53) . These cases which escape the rule ``one amino acid-one aminoacyl-tRNA synthetase'' are likely to reflect the capability of E. coli to adapt the activity of the synthetases to its environment in a yet unsuspected manner.

  
Table: Purification of LysS and LysU proteins


  
Table: Kinetic constants of LysS and LysU in the ATP-PP exchange, tRNA aminoacylation, and ApA synthesis reactions



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviation used is: LysRS, lysyl-tRNA synthetase.


ACKNOWLEDGEMENTS

We gratefully acknowledge F. Dardel for fruitful advice.


REFERENCES
  1. Hirshfield, I. N., Bloch, P. L., Van Bogelen, R. A., and Neidhardt, F. C.(1981) J. Bacteriol.146, 345-351 [Medline] [Order article via Infotrieve]
  2. Lévque, F., Plateau, P., Dessen, P., and Blanquet, S. (1990) Nucleic Acids Res.18, 305-312 [Abstract]
  3. Hirshfield, I. N., and Buklad, N. E.(1973) J. Bacteriol.113, 167-177 [Medline] [Order article via Infotrieve]
  4. Buklad, N. E., Sanborn, D., and Hirshfield, I. N.(1973) J. Bacteriol.116, 1477-1478 [Medline] [Order article via Infotrieve]
  5. Hirshfield, I. N., Yeh, F. M., and Sawyer, L. E.(1975) Proc. Natl. Acad. Sci. U. S. A.72, 1364-1367 [Abstract]
  6. Lévque, F., Gazeau, M., Fromant, M., Blanquet, S., and Plateau, P.(1991) J. Bacteriol.173, 7903-7910 [Medline] [Order article via Infotrieve]
  7. Neidhardt, F. C., and VanBogelen, R. A.(1981) Biochem. Biophys. Res. Commun.100, 894-900 [Medline] [Order article via Infotrieve]
  8. Hickey, E. W., and Hirshfield, I. N.(1990) Appl. Environ. Microbiol.56, 1038-1045 [Medline] [Order article via Infotrieve]
  9. Hassani, M., Pincus, D. H., Bennett, G. N., and Hirshfield, I. N. (1992) Appl. Environ. Microbiol.58, 2704-2707 [Abstract]
  10. Gazeau, M., Delort, F., Dessen, P., Blanquet, S., and Plateau, P. (1992) FEBS Lett.300, 254-258 [CrossRef][Medline] [Order article via Infotrieve]
  11. Lin, R., Ernsting, B., Hirshfield, I. N., Matthews, R. G., Neidhardt, F. C., Clark, R. L., and Newman, E. B.(1992) J. Bacteriol.174, 2779-2784 [Abstract]
  12. Ito, K., Kawakami, K., and Nakamura, Y.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 302-306 [Abstract]
  13. Newman, E. B., D'Ari, R., and Lin, R. T.(1992) Cell68, 617-619 [Medline] [Order article via Infotrieve]
  14. Willins, D. A., Ryan, C. W., Platko, J. V., and Calvo, J. M.(1991) J. Biol. Chem.266, 10768-10774 [Abstract/Free Full Text]
  15. Kawakami, K., Jönsson, Y. H., Björk, G. R., Ikeda, H., and Nakamura, Y.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 5620-5624 [Abstract]
  16. Craigen, W. J., Cook, R. G., Tate, W. P., and Caskey, T.(1985) Proc. Natl. Acad. Sci. U. S. A.82, 3616-3620 [Abstract]
  17. Hirshfield, I. N., Tenreiro, R., VanBogelen, R. A., and Neidhardt, F. C.(1984) J. Bacteriol.158, 615-620 [Medline] [Order article via Infotrieve]
  18. Clark, R. L., and Neidhardt, F. C.(1990) J. Bacteriol.172, 3237-3243 [Medline] [Order article via Infotrieve]
  19. Hassani, M., Saluta, M. V., Bennett, G. N., and Hirshfield, I. N. (1991) J. Bacteriol.173, 1965-1970 [Medline] [Order article via Infotrieve]
  20. Kawakami, K., Naito, S., Inoue, N., Nakamura, Y., Ikeda, H., and Uchida, H.(1989) Mol. & Gen. Genet.219, 333-340
  21. Furano, A.(1977) J. Biol. Chem.252, 2154-2157 [Abstract]
  22. Smith, D. K., Kassam, T., Singh, B., and Elliott, J. F.(1992) J. Bacteriol.174, 5820-5826 [Abstract]
  23. Coulondre, C., and Miller, J. H.(1977) J. Mol. Biol.117, 525-575 [Medline] [Order article via Infotrieve]
  24. Brevet, A., Chen, J., Lévque, F., Plateau, P., and Blanquet, S.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 8275-8279 [Abstract]
  25. Lawrence, F., Blanquet, S., Poiret, M., Robert-Gero, M., and Waller, J. P.(1973) Eur. J. Biochem.36, 234-243 [Medline] [Order article via Infotrieve]
  26. Blanquet, S., Fayat, G., and Waller, J. P.(1974) Eur. J. Biochem.44, 343-351 [Medline] [Order article via Infotrieve]
  27. Dardel, F.(1994) Comput. Appl. Biosci.10, 273-275 [Abstract]
  28. Miller, J. H.(1992) Experiments in Molecular Genetics, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  29. Kisselev, L. L., and Baturina, I. D.(1972) FEBS Lett.22, 231-237 [CrossRef][Medline] [Order article via Infotrieve]
  30. Rymo, L., Lundvik, L., and Lagerkvist, U.(1972) J. Biol. Chem.247,3888-3899 [Abstract/Free Full Text]
  31. Plateau, P., Gueron, M., and Blanquet, S.(1981) Biochimie (Paris) 63,827-830 [Medline] [Order article via Infotrieve]
  32. Charlier, J., and Sanchez, R.(1987) Biochem. J.248, 43-51 [Medline] [Order article via Infotrieve]
  33. Cole, F. X., and Schimmel, P. R.(1970) Biochemistry9, 480-489 [Medline] [Order article via Infotrieve]
  34. Garrison, P. N., and Barnes, L. D.(1992) in ApA and Other Dinucleoside Polyphosphates (McLennan, A. G., ed) pp. 29-61, CRC Press, Boca Raton, FL
  35. Plateau, P., and Blanquet, S.(1992) in ApA and Other Dinucleoside Polyphosphates (McLennan, A. G., ed) pp. 63-79, CRC Press, Boca Raton, FL
  36. Kitzler, J. W., Farr, S. B., and Ames, B. N.(1992) in ApA and Other Dinucleoside Polyphosphates (McLennan, A. G., ed) pp. 135-149, CRC Press, Boca Raton, FL
  37. Hirshfield, I. N., and Yeh, F. M.(1976) Biochim. Biophys. Acta435, 306-314 [Medline] [Order article via Infotrieve]
  38. Chakraburtty, K., Steinschneider, A., Case, R. V., and Mehler, A. H. (1975) Nucleic Acids Res.2, 2069-2075 [Abstract]
  39. Komine, Y., Adachi, T., Inokuchi, H., and Ozeki, H.(1990) J. Mol. Biol.212, 579-598 [Medline] [Order article via Infotrieve]
  40. Wittwer, A. J., and Stadtman, T. C.(1986) Arch. Biochem. Biophys.248, 540-550 [Medline] [Order article via Infotrieve]
  41. Meng, S. Y., and Bennett, G. N.(1992) J. Bacteriol.174, 2659-2669 [Abstract]
  42. Igarashi, K., Kashiwagi, K., Hamasaki, H., Miura, A., Kakegawa, T., Hirose, S., and Matsuzaki, S.(1986) J. Bacteriol.166, 128-134 [Medline] [Order article via Infotrieve]
  43. Kashiwagi, K., and Igarashi, K.(1988) J. Bacteriol.170, 3131-3135 [Medline] [Order article via Infotrieve]
  44. Tabor, H., Hafner, E. W., and Tabor, C. W.(1980) J. Bacteriol.144, 952-956 [Medline] [Order article via Infotrieve]
  45. Tabor, C. W., and Tabor, H.(1985) Microbiol. Rev.49, 81-99
  46. Auger, E. A., Redding, K. E., Plumb, T., Childs, L. C., Meng, S. Y., and Bennett, G. N.(1989) Mol. Microbiol.3, 609-620 [Medline] [Order article via Infotrieve]
  47. Ito, K., Oshima, T., Mizuno, T., and Nakamura, Y.(1994) J. Bacteriol.176, 7383-7386 [Abstract]
  48. Shi, X., Waasdorp, B. C., and Bennett, G. N.(1993) J. Bacteriol.175,1182-1186 [Abstract]
  49. Steffes, C., Ellis, J., Wu, J., and Rosen, B. P.(1992) J. Bacteriol.174,3242-3249 [Abstract]
  50. Watson, N., Dunyak, D. S., Rosey, E. L., Slonczewski, J. L., and Olson, E. R.(1992) J. Bacteriol.174, 530-540 [Abstract]
  51. Hirshfield, I. N., and Zamecnik, P. C.(1972) Biochim. Biophys. Acta259, 330-343 [Medline] [Order article via Infotrieve]
  52. Fromant, M., Fayat, G., Laufer, P., and Blanquet, S.(1981) Biochimie (Paris) 63, 541-553 [Medline] [Order article via Infotrieve]
  53. Wada, M., Sekine, K., and Itikawa, H.(1986) J. Bacteriol.168, 213-220 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.