Vol. 123, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Aug. 1975, p. 580-588 Copyright © 1975 American Society for Microbiology

Enhanced Level and Metabolic Regulation of Methionyl-Transfer Ribonucleic Acid Synthetase in Different Strains of Escherichia coli K-12 D. CASSIO,1 Y. MATHIEN, AND J. P. WALLER2 Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, 91190 Gif-sur- Yvette, France

Received for publication 6 March 1975

The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of' this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twof'old higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of' this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium. From Escherichia coli K-12 strains lysogenic for phage P2 in integration site H (27), histidinerequiring eductants, which contain deletions extending through the his operon into adjacent genes, have been isolated (26). One end of the deletion, the P2 prophage end located at 38.5 min, appears to be the same for all eductants. The other end, however, can be located to the lef't of the his operon, before, within, and after the mglP locus, near the methionyl-transfer ribonucleic acid (tRNA) synthetase structural gene (6). Since methionyl-tRNA synthetase is indispensable for protein biosynthesis, it ap' Present address: Centre de

Genetique Moleulaire,

pears certain that the eductants have this enzyme. However, it was considered that some P2-mediated deletions might cover a part of the structural gene of this enzyme or more likely the loci involved in the regulation of its synthesis. The former possibility was envisaged in view of the fact that earlier studies of this laboratory (8) have shown that 25% of' the protein can be removed in vitro by proteolysis without significantly affecting enzymatic activity. To test these hypotheses, the methionyl-tRNA synthetase of three independent eductants derived from strain AB311 was investigated. No structural alteration of' the enzyme was observed in the three eductants examined, but two of them exhibit a constitutively increased level of methionyl-tRNA synthetase. From recent studies, it appears that the

Cen-

tre National de la Recherche Scientifique, 91190 Gif-sur-

Yvette, France. 2Present address: Laboratoire de Biochimie, Ecole Polytechnique, 91120 Palaiseau, France.

580

VOL. 123, 1975

formation of some aminoacyl-tRNA synthetases is controlled not only by a repression-like mechanism mediated by the cognate amino acid or a derivative of it, but also by an additional process referred to as metabolic regulation, which is reflected by the coupling of synthetase formation to growth rate (23, 24). In this paper we describe how the specific activity of methionyl-tRNA synthetase varies with growth rate in different strains of E. coli K-12, particularly in strains that, because of their enhanced level of methionyl-tRNA synthetase, can be considered as mutants with altered regulation of this enzyme. These studies were carried out to verify whether methionyl-tRNA synthetase, like other synthetases of E. coli K-12, is subject to metabolic regulation and also to learn how the two processes of "repression" and "metabolic regulation" interact to control formation of the enzyme. In addition, we report that the level of methionyl-tRNA synthetase in a strain diploid for the structural gene of this enzyme varies with the phosphate concentration in the culture medium used. Conversely, no change in the level of methionyl-tRNA synthetase was observed in the case of a haploid strain grown in the presence of different concentrations of phosphate.

METHIONYL-tRNA IN E. COLI K-12. I.

581

disodium salt, and 0.4% glucose. In addition, two richer media were used that consisted of the basal medium plus glucose supplemented with 1.5% Casamino Acids or 1% tryptone (Difco) and 0.5% yeast extract (24). Additional enriched media used were 0.8% nutrient broth (6) and minimal medium 63 (11) containing thiamine at 0.5 mg/ml, 0.4% glucose, and 1% yeast extract (Difco). The cultures were incubated at 37 C with shaking and cells were harvested in exponential phase (absorbance = 1.5). Strains HfrH and EM20031 were also grown in minimal media 63 and MOPS containing 0.4% glucose with different concentrations of potassium phosphate as indicated in the text. No difference in growth rate was observed over the range of phosphate concentrations used. Growth rate was expressed as the reciprocal of the generation time. Preparation of cell extracts. Cells (0.5 to 1.0 g, wet weight) were suspended in 7 ml of 20 mM potassium phosphate buffer at pH 7.6 containing 10 mM 2-mercaptoethanol and subjected to sonic oscillation (10-kHz Raytheon) for 15 min at 0 C. The sonically disrupted extract was centrifuged at 16,000 x g for 20 min at 0 C and the supernatant fluid was dialyzed overnight against 2 liters of the same buffer. The protein content was determined colorimetrically by the method of Lowry et al. (17). Enzyme measurements. All measurements of enzymatic activity were performed on dialyzed crude extracts. The basal level (noninduced by D-serine) of D-serine deaminase was measured by the method described by McFall (18). The activity of the aminoacyl-tRNA synthetases studied was usually measured MATERIALS AND METHODS by the rate of amino acyl-tRNA formation as deBacterial strains. Six strains of E. coli K-12 were scribed previously (15). One unit of enzyme activity is used in this study. The strain used as reference was defined as that amount producing aminoacylation of 1 HfrH (thi-1) (3). Strain AB311 (thr-1-leu-6-thi-1-str- nmol of tRNA in 10 min at 30 C. In some cases the 8) and the P2-mediated eductants ed2, edlO, and edil aminoacyl-tRNA synthetases were tested by the derived from this strain were kindly provided by B. amino acid-dependent adenosine triphosphate (ATP) Rotman. These eductants were isolated as histidine- = PP1 exchange as described earlier (15), except that requiring strains and correspond, respectively, to the concentration of MgCl2 was 7 mM. One unit of strains QE76, QE84, and QE85 described by Sunshine enzyme activity is defined as the amount forming 1 and Kelly (26). The prototrophic merodiploid strain Mm of [32P]ATP in 15 min at 37 C. EM20031, which carries the episome F32 (19), was Apparent molecular weight determination. This obtained from E. McFall. determination was performed by filtration through a Media and methods of cultivation. For the char- calibrated Sephadex G-200 column according to Anacterization of methionyl-tRNA synthetase in the drews (1). different strains examined, cells were grown in miniPreparation of serum directed against methiomal medium 63 (11) containing thiamine at 0.5 nyl-tRNA synthetase. Rabbits were immunized by a mg/liter with 0.4% glucose as the carbon source and primary subcutaneous injection of 1 mg of homogenesupplemented with required growth factors at 0.5 ous methionyl-tRNA synthetase purified from strain mM. The cultures were incubated at 37 C with EM20031 (7), followed by two additional injections of shaking. Growth was monitored turbidimetrically by 1 mg of enzyme 3 and 6 weeks later, respectively. The measuring the absorbance at 420 nm. Cells were unfractionated serum was stored at -15 C and samharvested at 75% logarithmic phase (absorbance = ples were dialyzed against 20 mM potassium phos2.5). For studying the coupling of aminoacyl-tRNA phate buffer at pH 7.5 before use. Materials. The chromatographically homogeneous synthetase formation to growth rate, the different strains were grown in a minimal medium (designated L-amino acids used in the enzyme assays were purMOPS) composed of potassium morpholinopropane chased from Schwartz-Mann Research Laboratories. sulfonate exactly as described by Parker and Neid- Sodium [32P ]pyrophosphate and purified "4C-labeled hardt (24). The medium was supplemented with L-amino acids were obtained from the Commissariat a required growth factors at 0.5 mM. Carbon sources l'Energie Atomique (Saclay, France). D-Serine was used were 0.4% potassium acetate, 0.4% succinic acid purchased from the California Foundation for Bio-

582

CASSIO, MATHIEN, AND WALLER

chemical Research. Unfractionated tRNA was prepared from E. coli K-12 strain EM20031 according to Zubay (29) as described previously (4).

RESULTS Characterization of methionyl-tRNA synthetase in P2-mediated eductants derived from strain AB311. The specific activities of several aminoacyl-tRNA synthetases were determined in three P2-mediated independent eductants, in their parental strain as well as in the reference strain, HfrH (Table 1). Of the four aminoacyl-tRNA synthetases tested, all but methionyl-tRNA synthetase exhibited similar activities in all strains examined. We have previously reported that strain AB311 is characterized by a constitutive threefold increased level of methionyl-tRNA synthetase (6) that is probably due to an increase in net synthesis of the enzyme (9). The molecular event or TABLE 1. Level offour aminoacyl-tRNA synthetases in P2-mediated eductants from strain AB311a

J. BACTERIOL.

the selecting agent responsible for this increase is unknown. In two of the three eductants examined, the activity of methionyl-tRNA synthetase was higher than in the parental strain and attained seven times the usual level, as measured by the methioninedependent ATP-PP, exchange assay or the rate of methionyl-tRNA formation. The enhancement of methionyl-tRNA synthetase activity in eductants 2 and 10 could result from the production of an enzyme with higher specific activity or from an increase in synthesis of unaltered enzyme. To distinguish between these two alternatives, the neutralization curves of methionyl-tRNA synthetase activity of each strain by antibodies directed against purified enzyme were determined (Fig. 1). The activities of methionyl-tRNA synthetase in crude extracts from strains AB311, ed2, edlO, and edll were, respectively, three-, seven-, seven- and threefold higher than in HfrH and required, respectively, three, seven, seven, and

ATP = [l2P JPP, exchange

Strain

Methionyl-

Isoleucyl-tRNA

Leucyl-tRNA

synthetAse synthetase

synthetase

synthetase

Sp act

(U/mg of protein)

HfrH AB311 ed2 edlO edll

Sp act Sp act Rel- (U/mg Rel- (U/mg Relof ative of ative ative sp actb pro- sp actb pro- sp act' tein) tein)

1.87 (1.00) 6.05 3.24 13.20 7.06 12.50 6.68 6.00 3.21

3.90 4.40 3.65 3.30 3.40

(1.00) 1.12 0.94 0.85 0.87

6.60 6.43 6. 17 5.35 5.60

(1.00) 0.97 0.93 0.81 0.85

Aminoacyl-tRNA formation Strain

HfrH AB311 ed2 edlO edll

Methionyl-tRNA synthetase

Valyl-tRNA synthetase

Sp act (U/mg of protein)

Relative splati pac5

Sp act (U/lg of

20.9 63.2 147.0 156.0 69.4

(1.00) 3.02 7.03 7.46 3.32

90.0 92.4 90.7 86.4 87.7

protein)

Relative

Sp act' (1.00) 1.03 1.01 0.96 0.97

a Strains were grown in minimal medium 63 as described in Material and Methods. In these experiments growth rate values were 0.65, 0.70, 0.50, 0.35, and 0.60 generation/h for strains HfrH, AB311, ed2, edlO, and edll, respectively. I Relative to HfrH.

I

40

60

concentration or antisgrum(;ig protein/ml )

80

FIG. 1. Neutralization of methionyl-tRNA synthetase activity in crude extracts of P2-mediated eductants from strain AB311. Crude extracts from strains HfrH, AB311, ed2, edlO, and edll (each at a protein concentration of 65 jug/ml in 20 mM potassium phosphate buffer at pH 7.6 containing 200 ,g of bovine serum albumin/ml and 5 mM 2-mercaptoethanol) were incubated at 4 C for 5 h with varying amounts of antiserum prepared against purified methionyl-tRNA synthetase from strain EM20031. Samples were removed and assayed for methionyl-tRNA formation after the appropriate dilution. Methionyl-tRNA synthetase activity from a crude extract of HfrH (0), AB31I (0), ed2 (A), edlO (A), and edlI (0).

VOL. 123, 1975

METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. I.

three times as much antibody for neutralization compared with an extract from strain HfrH examined at the same protein concentration. These results are in agreement with an increased cellular level of unaltered methionyl-tRNA synthetase. To verify further this interpretation, the properties of methionyl-tRNA synthetase from ed2 and from its parental strain AB311 were compared. Strain ed2 was chosen rather than edlO because of its higher growth rate. The enzymes from both strains had similar affinities toward ATP and methionine; the Vmax value of the enzyme preparation from the eductant, calculated at saturation of all the substrates, was increased in proportion to the increase in its specific activity (Table 2). In addition, the two enzymes investigated showed the same magnesium ion requirement for the two reactions catalyzed by methionyl-tRNA synthetase. The molecular weight of the enzyme from strains HfrH, AB311, ed2, edlO, and edll was investigated by gel filtration through Sephadex G-200. In each case, the enzyme activity was eluted as a single component, the elution volume of which corresponded to an apparent molecular weight of 175,000. Moreover, we had previously shown that during incubation at 37 C of crude extract of strain HfrH, methionyl-tRNA synthetase is irreversibly modified by a protease into an enzymatically active form with an apparent molecular weight 60,000 (8). It was verified that the same modification also occurred in crude extracts from strains AB311, ed2, and edlO; the rate of modification was similar in each case. Metabolic regulation of methionyl-tRNA synthetase in various strains of E. coli K-12. To determine whether methionyl-tRNA synthetase is subject to metabolic regulation, the level of this enzyme was measured over a wide range of steady-state growth rates. It was of particular interest to extend these studies to strains such as AB311 and ed2, which have enhanced levels of this enzyme. In these experiments, valylTABLE 2. Kinetic parameters of methionyl-tRNA synthetase in crude extracts of strains AB 311 and ed2a

K., Source of

Sp actof

of enye(U/mg protein)

AB311 ed2

70.4 159.3

VA.ax Methi(U/mg of AP

(mM)

onine

protein)

0.17 0.17

3.7 4.0

91.5 206.2

aThese kinetic parameters were determined for the tRNA aminoacylation reaction.

583

tRNA synthetase activity was also determined as a control since, as shown earlier (23, 24), the level of this enzyme is clearly dependent on growth rate. For each strain examined, the valyl-tRNA synthetase activity varied with growth rate, as expected; moreover the methionyl-tRNA synthetase activity was also affected, but to a lesser extent (Fig. 2). Over a fivefold increase of growth rate, the levels of valyl-tRNA synthetase and of methionyl-tRNA synthetase were found to increase 2.2- and 1.8-fold, respectively. It should be pointed out that in contrast to an earlier report (24), the relation between aminoacyl-tRNA synthetase level and growth rate was not maintained when strains were grown in very enriched media such as nutrient broth (6) or the enriched medium (MOPSTGYE) of Parker and Neidhardt (24). In these cases, as shown in Table 3 for strain HfrH, a net decrease of methionyl- and valyl-tRNA synthetase activities was observed. The extent of this decrease depended on the strain examined. This discrepancy may be related to the observation (results not shown) that in strain ed2 grown in nutrient broth, the specific activity of methionyl-tRNA synthetase decreased progressively during the exponential phase, in contrast to its constancy during exponential growth in minimal media. In Fig. 3, the preceding data are expressed not

150E

.5

z

50-

3-.

5

0.5

1.0

1.5

groth rate(generation/hr)

FIG. 2. Level of methionyl- and valyl-tRNA synthetases in some strains of E. coli K-12 grown at different rates. Strains were grown in MOPS minimal medium with different carbon sources as described in the text in order to allow balanced growth at different rates. Methionyl- and valyl-tRNA synthetase activities were determined by the rate of aminoacyltRNA formation. The activities in strain HfrH (0), AB311 (0), and ed2 (A) are plotted as a function of growth rate. The results presented also include data obtained in minimal medium 63 containing glucose as carbon source.

584

CASSIO, MATHIEN, AND WALLER

TABLE 3. Level of methionyl- and valyl-tRNA synthetases in strain HfrH grown in various enriched mediaa Sp act (U/mg of protein) Growth

Medium

rate

Methi-

(genera-

onyl-

tion/h)

tRNA synthetase

MOPS-glucose1.33 25.7 Casamino Acids Nutrient broth 1.33 14.6 MOPS-tryptone1.80 22.6 glucose-yeast extract M63-glucose-yeast 1.80 14.7 extract a Grown as described in the text.

ValyltRNA synthetase

142.0 94.0 81.9 -

in terms of growth rate but in terms of culture medium; this representation allows a comparison of the methionvl- and valyl-tRNA synthetase activities of' strains HfrH, AB311, and ed2 grown in four different media. It appears that, although the levels of these two enzymes depend on growth rate and consequently on growth medium, for a given medium the levels of methionyl-tRNA synthetase in strains AB311 and ed2 were, respectively, three- and sevenfold higher than that of strain HfrH whereas the level of valyl-tRNA synthetase was similar in all three strains. Thus, the increase in methionyl-tRNA synthetase level observed in these strains was independent of growth conditions. Variation of methionyl-tRNA synthetase level on the merodiploid strain EM20031 as a function of phosphate concentration of the culture medium. Methionyl-tRNA synthetase purified from the merodiploid strain EM20031 is the subject of extensive investigations bearing on its primary structure (5), its tertiary structure (20), and its mechanism of action (4, 13). Strain EM20031 carries the episome F32 and is presumed to be diploid for the structural gene of methionyl-tRNA synthetase. We have previously reported that this strain is characterized by a fourfold increased level of the enzyme regardless of growth conditions (6). Moreover, it was found that the female parent of strain EM20031 has a normal level of methionyltRNA synthetase, whereas the Hfr AB311 from which the F32 episome originated has a threefold higher level of this enzyme. Thus the methionyl-tRNA synthetase activity of the merodiploid is equal to the sum of the activities of each parent (6). We have verified that methionyl-tRNA syn-

J. BACTERIOL.

thetase in strain EM20031 is subject to metabolic regulation as reported in the preceding section for the enzyme of strains HfrH, AB311, and ed2. However, when strain EM20031 was grown in MOPS medium, the level of methionyl-tRNA synthetase was found to be f'ive- to sixfold higher than that of strain HfrH, in contrast to the fourfold increase consistently observed in minimal medium 63. The only major difference in the composition of the two media is the phosphate concentration, which is 1.27 mM in MOPS medium compared with 100 mM in medium 63 (11). To assess the eff'ect of phosphate concentration on enzyme level, methionyl-tRNA synthetase activity was measured in strain EM20031 grown in MOPS medium at different concentrations of phosphate. Moreover, since strain EM20031 is haploid for the structural gene of' valyl-tRNA synthetase and diploid for that of D-serine deaminase, the levels of these two enzymes in response to phosphate were determined as controls. As shown in Fig. 4A, in strain EM20031 the level of' methionyl-tRNA synthetase varied significatively with the phosphate concentration of the MOPS medium; the maximal level was obtained when the strain was grown in the presence of 25 mM phosphate and reached seven times the usual level found in HfrH. Similarly, the level of' D-serine deaminase depended on the phosphate concentration of the medium, whereas in these growth conditions the valyl-tRNA synthetase activity was unaffected. On the other hand (Fig. 4B), there was no variation of these three enzymatic activities in the haploid strain HfrH grown in MOPS minimal medium in the presence of different concentrations of phosphate. Finally, the effect of phosphate concentration on the levels of methionyl-tRNA synthetase and D-serine deaminase from strain EM20031 was also observed by varying the phosphate concentration of minimal medium 63 (Fig. 5). In this case the highest level of these two enzymes was obtained at 10 mM phosphate.

DISCUSSION Recent studies indicate that synthesis of aminoacyl tRNA synthetases is regulated by a repression-like mechanism (for review, see reference 14). In a very few cases, regulatory mutants of these enzymes have been isolated and characterized. Paetz and Nass (22) have shown that the borrelidin resistance of the K-12-B borr3 strain is due to a constitutive fivefold increase of the level of threonyltRNA synthetase. Moreover, it was reported that about 30% of the borrelidin-resistant mutants of E. coli K-12B bear regulatory lesions

METHIONYL-tRNA IN E. COLI K-12. I.

VOL. 123, 1975

585

affecting threonyl-tRNA synthetase. This type of mutant is characterized by a four- to sixfold increased level of structurally unaltered threonyl-tRNA synthetase (21). Furthermore, Clarke et al. (10) have isolated an E. coli mutant in which the level of seryl-tRNA synthetase was five times that of the parental strain. This level was not affected by serine deprivation, in contrast to the response of the parental strain. It was found that this mutation, denoted serO, lies very close to the structural gene for seryl-tRNA synthetase and is cis dominant in a stable merodiploid. These results suggest that serO is an operator site involved in the control of the serS gene. In this report, we have investigated the methionyl-tRNA synthetase from E. coli K-12

._

t 2r,

E _ .0

Z

Z5

'IF

50 50

A

S

G

CA

FIG. 3. Level of methionyl- and valyl-tRNA synthetases in some strains of E. coli K-12 grown in different media. Straimq were grown as described in the text in MOPS minimat medium in the presence of 0.4% potassium acetate (A), 0.4% succinic acid (S), 0.4% glucose (G), or 0.4% glucose supplemented with 1.5% Casamino Acids (CA). The methionyl- and valyl-tRNA synthetase activities of strain HfrH (darkened area), AB311 (hatched area), and ed2 (light area) grown in these different media are presented. Growth rates of strain HfrH grown in A, S, G, and CA media were 0.26, 0.53, 0.70, and 1.33 generationlh, respectively. The corresponding values for strain AB311 were 0.26, 0.65, 0.83, and 1.25 generation per h,

150 200 10o 950 phosphate concentration of growth medium (mM)

FIG. 4. Level of D-serine deaminase and methionyl- and valyl-tRNA synthetases in the merodiploid strain EM20031 and in the haploid strain HfrH grown in MOPS minimal medium at different concentrations of phosphate. Both strains were grown at 37 C in MOPS minimal medium at different concentrations of phosphate in presence of 0.4% glucose. Cells were harvested in exponential phase and the level of D-serine deaminase (A) and valyl- (0) and methionyl-tRNA synthetases (0) in strains EM20031 (A) and HfrH (B) were measured. The relative specific activity of each enzyme is normalized to that of an extract from strain HfrH grown in minimal medium 63 containing 100 mM phosphate.

respectively. The corresponding values for strain ed2 were 0.19, 0.45, 0.66 and 0.91 generation/h, respectively.

586

CASSIO, MATHIEN, AND WALLER

71 6mz

-E

5.

0a,\

4.

1-4

3.

w

1-

1 00-O-o

8

50 100 phosphate concentration of growth medium(mM)

FIG. 5. Level of D-serine deaminase (A), methionyl-tRNA synthetase (a) and valyl-tRNA synthetase (0) in the merodiploid strain EM20031 grown in minimal medium 63 containing different concentrations of phosphate. The relative specific activity of each enzyme tested is normalized as described in Fig. 4.

strains carrying P2-mediated deletions located near the structural gene of' this enzyme. These eductants were isolated from strain AB311, which exhibits a constitutive threefold increased level of methionyl-tRNA synthetase. In two eductants (ed2 and edlO) the level of this enzyme is twofold higher than in their parental strain, regardless of growth conditions. By a number of criteria, including antigenic properties and molecular weight, the methionyl-tRNA synthetase from these two eductants is indistinguishable from that of the parental strain. From these results we conclude that the increased level of methionyl-tRNA synthetase observed in these strains is due not to the production of an altered enzyme but to an increase in the net synthesis of' normal enzyme. This overproduction of methionyl-tRNA synthetase is very likely related to the eduction of' P2. It appears that in none of the eductants examined does the P2-mediated deletion extend into the structural gene of' methionyl-tRNA synthetase. However, in some cases these deletions presumably induce modifications that affect the expression of this gene. This assertion is supported by the analysis of another series of independent eductants (result not shown) derived from E. coli K-12 strain S139 (25). We have found that, of f'ive of these eductants, three have altered levels of methionyl-tRNA synthetase. According to recent results (2; and Cassio, unpublished data), methionyl-tRNA is involved in repressional control of methionyl-tRNA synthetase. Thus, in strain HfrH. a decrease in the

J. BACTERIOL.

intracellular level of methionyl-tRNA induced by the specific inhibitor methioninyl adenylate (7) leads to derepression of the enzyme. In contrast, the methionyl-tRNA synthetase level in strains AB311 as well as ed2 fails to increase under the same conditions. This different behavior lends support to the assumption that the latter strains can be regarded as regulatory mutants. However, it will be necessary to undertake a genetic analysis of these strains to determine the precise nature of the regulatory element(s) altered. In the case of strain EM20031, which carries the F32 episome originating from strain AB311, it was shown that the high level of methionyl-tRNA synthetase that characterizes strain AB311 is still expressed (6). Thus the location of the mutation responsible for the high level of' methionyl-tRNA synthetase in strain AB311 could be in a cis acting regulatory locus for the structural gene of' methionyl-tRNA synthetase. Recently, Parker et al. (23, 24) reported that the rate of synthesis of some aminoacyltRNA synthetases is coupled with growth rate. In this report we show that methionyl-tRNA synthetase of E. coli K-12 is subject to such metabolic regulation. The specific activity of the enzyme was found to increase approximately twof'old over a fivefold increase in growth rate. Moreover, metabolic regulation governs methionyl-tRNA synthetase formation even in strains such as AB311 and ed2. in which the repression-like mechanism that controls the synthesis of this enzyme is altered. Consequently, the two processes that regulate the intracellular level of methionyl-tRNA synthetase appear to be independent or at least dissociable. Lastly, we have noted that in the merodiploid strain EM20031, the levels of methionyl-tRNA synthetase and D-serine deaminase vary with the phosphate concentration of growth medium, whereas the level of valyl-tRNA synthetase does not change. This observation is probably related to the f'act that this strain is diploid for the structural genes of' methionyl-tRNA synthetase and D-serine deaminase, whereas it is haploid for that of valyl-tRNA synthetase. Moreover, in the haploid strain HfrH there is no variation of these three enzymatic activities in response to phosphate concentration of growth medium. The effect of phosphate on the levels of methionyl-tRNA synthetase and D-serine deaminase in strain EM20031 was observed in two different minimal media. Both enzymes responded similarly to phosphate concentration. Highest levels were observed at 25 mM phosphate in MOPS medium and 10 mM phosphate in minimal

METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. I.

Vol. 123, 1975

medium 63; in addition, for each enzyme the maximal level attained was of the same order in both media. The variation of methionyl-tRNA synthetase activity in response to phosphate was larger than that of D-serine deaminase. This is attributed to the fact that the episome F32 originates from strain AB311, which has a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. The growth rate of strain EM20031 was unchanged throughout the range of phosphate concentrations used. Consequently, the phosphate-dependent variation of methionyl-tRNA synthetase cannot be ascribed to the process of metabolic regulation described earlier. The effect of phosphate on the levels of D-serine deaminase and methionyl-tRNA synthetase in strain EM20031 probably corresponds to a gene dosage effect. It is very likely indeed that the number of F32 episomes per chromosome in strain EM20031 varies to some degree depending on phosphate concentration. The number of copies of an F-prime factor per chromosome has been estimated to be between one and two, depending on the particular F prime used (16). In the case studied here, if one assumes that the episomal and chromosomal genes are expressed with the same efficiency, the ratio of episome to chromosome appears to vary between 0.9 to 1.7 over the range of phosphate concentrations used. The explanation for this variation may be related to the fact that the initiation of replication of an episome usually does not coincide with that for the chromosome (28) and occurs at different times of the division cycle, depending on growth conditions (12). ACKNOWLEDGMENTS We acknowledge B. Bachmann, B. Rotman, and M. G. Sunshine for kindly providing us with the E. coli K-12 strains. Ours thanks are given to M. Kaminski for preparation of the antiserum used in this work. We are most grateful to M. Weiss for critical reading of the manuscript. This work was supported in part by grants from the Delegation Generale a la Recherche Scientifique (project no. 71.7.3117) and the Commissariat a l'Energie Atomique (Saclay). LITERATURE CITED 1. Andrews, P. 1965. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96:595-606. 2. Archibold, E. R., and L. S. Williams. 1973. Regulation of methionyl-transfer ribonucleic acid synthetase formation in Escherichia coli and Salmonella typhimurium.

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Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12.

The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the struc...
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