Vol. 131, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Aug. 1977, p. 589-591 Copyright © 1977 American Society for Microbiology

Two Modes of Metabolic Regulation of Lysyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli K-121 IRVIN N. HIRSHFIELD,* CATHERINE LIU, AND FU-MEEI YEH The John Collins Warren Laboratories, Huntington Memorial Hospital of Harvard University, Massachusetts General Hospital, Boston, Massachusetts 02114

Received for publication 17 January 1977

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

While studying the lysyl-transfer ribonucleic acid (tRNA) synthetase (tRNA ligase [adenosine monophosphate], EC 6.1.1.6) from Escherichia coli K-12, mutants of the synthetase that showed singular properties were isolated (10, 11). These mutants had 2 to 5% of the wild-type lysyl-tRNA synthetase level when cultured in minimal medium but normal activity of the synthetase when grown in broth (10). Subsequent investigation of this observation led to the discovery that certain metabolites, alone or in combination, could replace the broth and restore the lysyl-tRNA synthetase activity in the mutants to normal levels (4, 8). These metabolites are L-alanine, L-alanine plus 1)-fructose (8), or, most potently, leucine dipeptides such as glycyl-L-leucine (3). These compounds will also stimulate lysyl-tRNA synthetase activity in wild-type E. coli K-12 (4, 8); thus, their effect in the mutant strains appears to be an amplification of events that can occur in wildtype strains. By several criteria the stimulation of the synthetase activity by the metabolites I Publication no. 1524 of the Cancer Commission of Harvard University.

has been shown to be the result of new enzyme synthesis (4, 8, 11). More recently it has been shown that the lysyl-tRNA synthetase purified from wild-type cells grown with 20 mM L-alanine or 3 mM glycyl-L-leucine has different physical and kinetic properties from the corresponding synthetase purified from cells grown only in minimal medium (9, 12). These results induced us to postulate that the metabolites could influence the properties of the enzyme by an unknown modification mechanism. By taking this cue, we examined the behavior of lysyl-tRNA synthetase in mutants defective in possible protein modification pathways. One well-documented mechanism for protein modification is that of methylation (25). A key enzyme in the methylation process is S-adenosyl-L-methionine (SAM) synthetase, which activates the methyl group of methionine to form S-adenosylmethionine. Mutants with 3% of the normal activity of SAM synthetase, metK mutants (7; E. W. Hafner, C. W. Tabor, and H. Tabor, Fed. Proc. 35:1547, 1976), were tested for lysyl-tRNA synthetase activity. The activ589

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ity of the synthetase was found to be elevated two- to fourfold in these mutants when compared with the appropriate isogenic parent. Upon further evaluation of one of these metK mutants, RG62, it was found that not only the activity, but also some of the properties, of the lysyl-tRNA synthetase are altered. In the past few years there has been welldocumented evidence for metabolic regulation of aminoacyl-tRNA synthetases in E. coli (19, 23, 26, 27). It has been of interest to us to determine if the regulation of lysyl-tRNA synthetase by metabolites such as glycyl-L-leucine or alanine is interrelated with the above type of metabolic regulation. The data presented in this manuscript clearly indicate that these two forms of metabolic regulation operate independently. MATERIALS AND METHODS Bacterial strains. The pertinent strains are listed in Table 1. Growth of strains. Cells were grown in minimal medium, as described by Davis and Mingioli (5), or in the MOPS (potassium morpholinopropane sulfonate) minimal medium described by Neidhardt et al. (22). Supplemented minimal medium was made according to the procedure of Novick and Maas (24). The amino acids histidine, arginine, and lysine were added at a final concentration of 0.01%. When the cells were grown in minimal medium in the presence of the peptide, glycyl-L-leucine, 0.75 mM Lisoleucine was added to prevent growth inhibition by the peptide (36). All experiments were conducted under aerobic conditions at 37°C, and the growth of the cells was measured on a Coleman Junior spectrophotometer. Cells grown in minimal medium were monitored at 490 nm, and those grown in supplemented medium were monitored at 580 nm. In all experiments the cells. were grown for at least four generations, and growth was terminated at an optical density of 0.2 at either wavelength. Preparation of crude enzyme extract. The crude enzyme extract was prepared as described previously (8). When the arginyl-, seryl-, and valyl-tRNA synthetases were being assayed, 10 mM dithiothreitol was included in the extract. Protein determination. Protein was determined TABLz 1. E. coli K-12 strains Strain Wild type RG62 RG109 RG314 RG317

EWH80 EWH64 Hfr H W3010 Q2 W3010 Q3

Relevant genotype metK+ rel+ metK84 rel+ metK86 rel+ lysA recA thi metK84 KLF-16 (IysA+ metK+)/ RG314 metK+ rel metK rel thi arg trp gly rel+ arg trp gly rel

R. R. R. R. R.

Source Greene Greene Greene Greene Greene

E. Hafner E. Hafner J. C. Patte P. Primakoff P. Primakoff

by the method of Lowry et al. (17) with crystalline bovine serum albumin (Sigma Chemical Co.) as a standard. Gel filtration chromatography experiments. Gel filtration chromatography has been described in detail in an earlier publication (12). The following modifications were made in the experiments presented here. The cells were grown only to an optical density of 0.2 at 490 nm. The serine protease inhibitor diisopropylfluorophosphate was added to the sonic extract buffer at a concentration of 1.5 mM before the sonic extract was prepared. The flow rate on the Sephadex G-200 column was increased from 3.4 to 4.4 to 4.8 ml/h, and 140 fractions of 1.1 to 1.2 ml were collected. As before, the internal standards used in these experiments were catalase, 240,000 daltons, lactate dehydrogenase, 135,000 daltons, and cytochrome c, 12,400 daltons (1). Cytochrome c was measured spectrophotometrically at 410 nm (1). Catalase activity was assayed by measuring the decomposition of H202 at 240 nm (2), and lactate dehydrogenase was assayed by measuring the oxidation of reduced nicotinamide adenine dinucleotide at 340 nm (15). Aminoacyl-tRNA synthetase assays. The activity of the lysyl-tRNA synthetase was assayed at pH 7.8 as described by Hirshfield et al. (10, 11). One microgram of crude extract protein was used in these assays. Arginyl-, seryl-, and valyl-tRNA synthetases were assayed in the same manner as the lysyl-tRNA synthetase with the following exceptions. The buffer used was 0.1 M tris(hydroxymethyl)aminomethane (Tris)-maleate, pH 7.25. Three micrograms of crude extract protein was used in these assays. A sulfhydryl-reducing agent, dithiothreitol, was added to the reaction mixture at a final concentration of 2 mM. Finally, the specific activity of these L-14C-amino acids was 50 Ci/mol. The units of activity for the synthetases were calculated on the basis of the nanomoles of 14C-amino acid incorporated into tRNA in 10 min. Specific activity is defined as units per milligram of protein. Heat inactivation studies. Heat inactivation studies were conducted in two ways. In one set of experiments the various lysyl-tRNA synthetase preparations were preincubated at 43°C for 15 s. The preincubation was conducted at pH 7.8 (0.1 M Tris). No substrates (adenosine 5'-triphosphate, lysine, or tRNA) were present in the preincubation. After the preincubation, the enzyme was placed in ice at 0 to 4°C, and the substrates were added. The remaining enzyme activity was assayed at 30°C, pH 7.8 (0.1 M Tris), for 3 min. As a control, the activity of the lysyl-tRNA synthetase was determined at 30°C without prior preincubation. A second method used to test for thermal inactivation was to incubate the lysyl-tRNA synthetase preparations at 36 and 43°C in the presence of all substrates for 3 min at pH 7.8 (0.1 M Tris). The activity at 43°C was compared to the activity at 360C. Materials. The uniformly labeled amino acids were purchased from either the New England Nuclear Corp., Boston, Mass., or Schwarz/Mann, Orangeburg, N.J. Sephadex G-200 was obtained from Pharmacia Fine Chemicals, Inc., Piscataway, N.J.

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Cytochrome c was obtained from Sigma Chemical Co., St. Louis, Mo.; lactate dehydrogenase (band 5, native) was obtained from Boehringer Mannheim, Corp., Indianapolis, Ind.; and catalase was obtained from Worthington Biochemicals Corp., Freehold, N.J.

RESULTS Lysyl-tRNA synthetase activity in metK mutants. Recent work from this laboratory has shown that the lysyl-tRNA synthetase from wild-type cells cultured in glucose minimal medium in the presence of 3 mM glycyl-L-leucine has different physical and kinetic properties from the corresponding synthetase from cells grown only in minimal medium (9, 12). These changes include greater thermostability, increased resistance to proteolysis, and differences in the K. values for lysine and tRNA'YS (9, 12). These results prompted us to propose that two forms of the lysyl-tRNA synthetase exist, and that one enzyme form may be derived from the other by an unknown post-translational modification mechanism (9, 12). To approach this problem we devised the strategy of testing the effect of glycyl-L-leucine on the synthetase activity in mutants of E. coli known to be defective in possible protein modification pathways. In the initial experiments, a mutant that had a deletion for both adenyl cyclase and the cyclic adenosine 3',5'-monophosphate-binding protein, eliminating cyclic adenosine monophosphate from the cell (30), and a mutant defective in the leucyl, phenylalanyl-tRNA-protein transferase (31) were examined. These experiments were negative in that the peptide (3 mM) elicited the normal stimulation of lysyl-tRNA synthetase activity (two- to threefold). Mutants with 3% of the SAM synthetase activity (7; Hafner et al., Fed. Proc. 35:1547, 1976) were then analyzed with encouraging results. In these experiments the activity of the lysyl-tRNA synthetase was elevated two- to fourfold even in the absence of the peptide (Table 2). The results were the same irrespective of whether the carbon source was acetate or glucose. Neither did it matter whether the metK strains were stringent (RG62, RG109) or relaxed (EWH64). Addition of 3 mM glycyl-L-leucine to the medium resulted in little or no increase in lysyl-tRNA synthetase activity (Table 2) in these mutants. Heat inactivation experiments. The above results were indicative of an effect of methylation on the activity of lysyl-tRNA synthetase. However, the specter of a second mutation that could independently influence the synthetic rate of the synthetase was present. It seemed unlikely that this would happen in three inde-

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pendently isolated spontaneous metK mutants, but we deemed that it would be useful to demonstrate that changes in the physical properties of lysyl-tRNA synthetase had occurred. If the metK mutation accomplished the same end result as adding the peptide to the growth medium, then it would be expected that, in addition to an increase in activity, the lysyl-tRNA synthetase from a metK strain should be more heat stable when compared with the enzyme from its isogenic parent. We therefore grew the wild-type strain in acetate minimal medium with or without 3 mM glycyl-L-leucine and the metK mutant (RG62) in acetate minimal medium. By using crude extracts, we compared the heat stability of the lysyl-tRNA synthetase in these three preparations. In the first set of experiments (Table 3), the inherent thermostability of the enzymes was examined by preincubation at 43°C in the absence of substrates. The loss of activity at 43°C under these conditions is rapid, and 15 s was the shortest practical time that we could use to conduct this assay. Increasing the preincubation time to 30 or 60 s had no influence on the results. In these experiments, it was found that the lysyl-tRNA synthetase from the metK mutant was more thermostable than the corresponding synthetase from its parent strain and equally stable to the lysyl-tRNA synthetase from wild-type cells grown with the TABLE 2. Activity of lysyl-tRNA synthetase in Sadenosyl-L-methionine synthetase mutant strains Strain

Carbon

source a

Wild type Wild type RG62 RG62 RG109 RG109

Glucose Glucose Glucose Glucose Glucose Glucose

Wild type Wild type RG62 RG62

Acetate Acetate Acetate Acetate

Glycyl-L-leuLysyl-tRNA synthetase cineb

(U/mg) 38

+

+ + + +

133 129 114 90 107 26 86 78 71

18 Glucose EWH80C + 52 EWH80 Glucose EWH64 37 Glucose EWH64 Glucose + 30 a Cells were grown in the minimal medium of Davis and Mingioli (5). The carbon sources were present at a concentration of 0.4%. b Glycyl-L-leucine was added to the medium at a concentration of 3 mM. + Indicates the presence of the peptide in the growth medium. c EWH80 is a metK+ strain and is the isogenic parent of EWH64 (metK).

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peptide. The synthetase from the metK mutant was more thermostable than its isogenic counterpart whether the amount ofprotein used was the same or adjusted to equalize the activity of the synthetases (Table 3, 0.5 ,ug for RG62 and 2.0 ,ug for wild type). The mechanism of the heat-induced loss of activity of the synthetase is not presently understood. Work with purified lysyl-tRNA synthetase suggests that there is a stronger subunit interaction in the enzyme form from peptide-grown cells, and, thus, diminished loss of activity of this form upon heating may reflect a lower rate of dissociation of an active dimer into inactive monomers (I. N. Hirshfield and F.-M. Yeh, unpublished data). In a second set of experiments, the synthetases from the wild-type strain and RG62 were tested for their heat stability in the presence of all the substrates, a condition that generally will afford more protection against thermal inactivation due to binding of the substrates to the enzyme (Table 4). Although the heat stability of the wild-type synthetase did increase compared with the data in Table 3, it was still less stable than the lysyl-tRNA synthetase from the metK mutant RG62. Similar results are found with cells grown in glucose minimal medium. Gel filtration chromatography experiments. As another criterion for demonstrating an alteration in the properties of the lysyltRNA synthetase from the metK mutants, we chose to examine the molecular-weight profile

of the synthetase on Sephadex G-200. This method gives a qualitative evaluation of the action of endogenous proteases on the enzyme. We have previously shown that fewer molecular-weight species of the lysyl-tRNA synthetase are found when wild-type cells are grown in minimal medium with the peptide (12). We have been investigating the molecular weight of lysyl-tRNA synthetase as a separate issue and have found with several E. coli strains grown under different conditions that, invariably, the presence of the peptide in the growth medium results in a restriction of the number of molecular-weight species ofthe enzyme (I. N. Hirshfield, F.-M. Yeh, and T. N. McMillian, unpublished data). Often, but not always, this restriction results in a clustering of the species of lysyl-tRNA synthetase around a median of 120,000 to 130,000 daltons. The prediction derived from these experiments would suggest that lysyl-tRNA synthetase from a metK mutant should be less polymorphic than the synthetase from the parent strain when both were grown in glucose minimal medium in the absence of the peptide. The results presented in Fig. 1 show that this is precisely what was found. Moreover, the molecular-weight species of the synthetase from the metK mutant RG62 6r

a

LDH

CAT

4

TVA

-

TABLE 3. Effect of preincubation at 43°C on the activity of lysyl-tRNA synthetase

m

IM I

Amt of Stan pri protein Strain (pg) 1.0 Wild type 2.0 Wild type Wild type + 1.0

Sp act (U/mg) Activity after Preincu- No prein- preincubabateda cubation tion (%) 4.2 33.6 12.5 33.5 11.6 3.9 38.1

104.5

36.5

55.9 33.6

126.5 122.1

44.1 27.5

CC

M

I

-l

l

120

130

1.2

Gly-L-Leu RG62 RG62 a

1.0 0.5

Samples were preincubated at 43°C for 15 s as described

in the text.

80

90

100

110

140

FRACTION NUMBER

FIG. 1. Profile of apparent molecular weight of

TABLE 4. Effect of temperature on lysyl-tRNA synthetase activity in the presence of substratesa

a

70

Strain

Protein (,ug)

Ratio of sp act

Wild type RG62

1.0 1.0

0.48 1.00

Wild type RG62

0.5 2.0

0.44 0.96

(43"C/36"C)

Samples were incubated at pH 7.8 for 3 min.

lysyl-tRNA synthetase species on Sephadex G-200. (a) Wild-type (metK+) strain grown in minimal medium to early log phase (OD 0.2 at 490 nm). Apparent weights ofpeaks (in daltons) were: I, 250,000; II, 210,000; III, 200,000; IV, 160,000; V, 135,000; VI,

99,000; VII, 88,000; and VIII, 74,000. (b) The metK

mutant RG62 grown in minimal medium to OD 0.2. Apparent weights of peaks (in daltons) were: I, 145,000; II, 125,000; III, 110,000. A 20-pl portion of each wild-type fraction and 10 pi of each mutant fraction were assayed for lysyl-tRNA synthetase activity at pH 7.8 for 3 min.

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did cluster at about 120,000 to 130,000. The apparent molecular weights of the synthetase species from the wild-type cells range from 250,000 (I) to 75,000 (VIII) (see legend to Fig. 1). Experiments of our own (Hirshfield, Yeh, and McMillian, unpublished data) and others (6) presently suggest to us that the enzyme is a dimer of at least 180,000 to 190,000 daltons. Smaller forms would be produced by the proteolytic processing of the enzyme. At this time not enough information is available to discuss whether the small amount ofthe 250,000-dalton form is a dimer. This form has been observed in a number of experiments conducted in this laboratory with several E. coli strains (Hirshfield, Yeh, and McMillian, unpublished data). The results presented in this section, in addition to those of the heat inactivation experiments, strengthen the concept that the properties of lysyl-tRNA synthetase can be influenced by methylation, and considerably dampen the likelihood that the increase in synthetase activity in the metK mutant is due to a second mutation, which increases the amount of the synthetase. Lysyl-tRNA synthetase activity in a metK+/ metK merodiploid. Hunter et al. (13) have demonstrated that the metK+ allele is dominant in merodiploids of the constitution metK+/ metK. Analysis of the lysyl-tRNA synthetase activity in the merodiploid RG317 shows that the activity of the enzyme returns to the level found in the metK+ strain (Table 5). This result argues further in favor of the idea that the activity and properties of the lysyl-tRNA synthetase can be influenced by the metK gene. Possible relation between the mode of action of glycyl-L-leucine, and growth rate-mediated metabolic regulation. In the past few years, work by Neidhardt and his associates (19, 23, 26, 27) with E. coli has shown that the activity, and apparently the synthesis, of several aminoacyl-tRNA synthetases can be loosely coupled to the growth rate of the cell. This type of regulation has been termed "metabolic regulation." It has been of interest to know whether this growth rate-related regulation is in any way linked to the regulation of lysyl-tRNA synthetase by metabolites such as

glycyl-L-leucine (11, 12). To analyze this potential relationship, cells were grown in MOPS minimal medium (22) with a poor carbon source (acetate) or supplemented with amino acids, vitamins, purines, and pyrimidines (SMM), in addition to glucose, to create an enriched medium. The generation time of the strain used in the first set of experiments (Hfr H; Table 6) was 150 min in acetate and 25 min in glucose-SMM. As an additional factor, 3 mM glycyl-ileucine was added to half of the cultures. MOPS minimal medium was used in these experiments to emulate as closely as possible the conditions of Parker and Neidhardt (27), and McKeever and Neidhardt (19). In the first set of experiments strain Hfr H was used because it had already been demonstrated that glycyl-ileucine could stimulate the lysyl- as well as the arginyltRNA synthetase in an enriched medium with this strain (4), and it was of interest to test other synthetases in this experiment. The intention of this experiment was to determine if the mechanism of action of the peptide and the mechanism of metabolic regulation might operate by some common mode. The data in Table 6 indicate that most likely these regulatory mechanisms act independently. First, the stimulation of lysyl-tRNA synthetase by the peptide is quantitatively similar whether the cells were grown in acetate or glucose-SMM. If the peptide acted in a manner similar to that of metabolic regulation then it would have been more likely to expect that the stimulation of the synthetase by the peptide would be greater in cells grown in acetate, for the activity of the synthetase is lower in that medium than in the enriched medium. A second crucial element in this analysis is the effect of the peptide on the synthetase activity in cells grown in the enriched medium. If the peptide and growth rate-related metabolic regulation worked through a common mechanism, then it would be unlikely that the peptide would stimTABLE 6. Influence of glycyl-L-leucine and growth medium on aminoacyl-tRNA synthetase activity in strain Hfr H Growth mediuma

TABLE 5. Lysyl-tRNA synthetase activity in

a

metK+/metK merodiploid Strain

Wild type RG314 RG317

Pertinent genotype

metK+ metK84

KLF16/metK84

Lysyl-tRNA synthetase (U/ mg) 32 64 33

593

Acetate Acetate + Gly-L-Leu Glucose-SMM Glucose-SMM + Gly-LLeu

Aminoacyl-tRNA synthetase (sp act) Lysine Arginine Serine Valine 12 28 50 120

11.9 12.3 42.5 80.0

4.1 7.5 14.5 22.0

26.0 27.5 78.8 116.0

a Carbon sources were present in the medium at concentrations of 0.4%. The peptide glycyl-ileucine was used at a concentration of 3 mM.

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ulate the level of lysyl-tRNA synthetase beyond that seen in the enriched medium, In this experiment, the synthetase level increased about fourfold by metabolic regulation (glucoseSMM/acetate); nevertheless, addition of the peptide (which does not increase the growth rate) raised the synthetase activity another two- to threefold. Similar results have been found with lysyl-tRNA synthetase with other strains of E. coli K-12. Studies on the arginyl-, valyl-, and seryltRNA synthetases showed that, as expected (19, 27), they all responded to growth-mediated metabolic regulation. When grown in glucoseSMM, all of these synthetases responded to the presence of glycyl-L-leucine, although the response was weaker than that of lysyl-tRNA synthetase. In acetate medium, only the seryltRNA synthetase showed any response to the presence of the peptide in the growth medium, and this was less than twofold. Influence of the metK mutation and growth medium on aminoacyl-tRNA synthetase activity. Since the experiments reported above have shown that the metK mutation can influence both the activity and properties of lysyltRNA synthetase, it was of interest to determine if the effect was pleiotropic. In the metK mutant RG62, lysyl-tRNA synthetase as usual displayed a two- to threefold elevation of activity, which was independent of the growth medium (Table 7). There was also an effect of the growth medium on the activity of this synthe-

tase in strain RG62 (Table 7). This latter result

again indicates that the growth rate-related regulatory mechanism is independent of the apparent involvement of methylation in the modulation of lysyl-tRNA synthetase activity. Of the other synthetases tested, only the seryltRNA synthetase showed any sign of being affected by the metK mutation. This response, although quite weak, was observed in four of five experiments with extracts from cells grown in acetate medium (Table 7). The presence of the metK mutation did not have any adverse effect on the response of these synthetases to the growth rate-related mechanism. Although not shown, 3 mM glycyl-Lleucine stimulated the lysyl- and seryl-tRNA synthetases in the wild-type strain grown in acetate medium to the same extent as that shown in Table 6, but, again, it had no effect on the arginyl- or valyltRNA synthetases in this medium. Influence of the rel gene on aminoacyltRNA synthetase activity. Whereas the effect of growth rate on the activity of the aminoacyltRNA synthetases appeared normal in metK+ rel+ strains, examination of this phenomenon in the metK+ rel strain EWH80 produced results that suggested the possibility that metabolic regulation might not be normal in this strain. However, since the isogenic rel+ parent of this particular strain was not available, it was not certain whether these results (Table 8) were peculiar to this strain. Therefore, the isogenic pair Q2 (rel+) and Q3 (rel) were grown

TABLE 7. Influence of the growth medium on aminoacyl-tRNA synthetase activity in a wild-type and metK mutant strain Aminoacyl-tRNA synthetase (sp act) Growth mediuma

Arginine

Lysine

WTb

MUT

WT

MUT

Serine WT

Valine MUT

WT

MUT

Acetate

25.4 74 26.3 28.8 5.8 7.5 16.2 15.0 Glucose-SMM 49.0 114 55.0 49.8 13.8 16.2 40.0 32.5 a The concentration of the carbon sources in the growth medium was 0.4%. b In these experiments the wild-type (WT) strain was supplied by R. Greene (Table 1) and the mutant (MUT) strain was RG62. The generation times for these strains are as follows: for wild type -acetate, 160 min, glucose-SMM, 25 min; and for RG62 -acetate, 200 min, glucose-SMM, 33 min.

TABLE 8. Influence of the relA gene on aminoacyl-tRNA synthetase activity Aminoacyl-tRNA synthetase (sp act) Growth mediUMa

Lysine

Arginine

Serine

Valine

EWH80b Q2 Q3 EWH80 Q2 Q3 EWH80 Q2 EWH80 Q2 Q3 Q3 Acetate 40 28 48 41 20 34 8.7 6.6 11.8 25 16 24 Glucose-SMM 60 90 98 54 48 45 12.5 21.9 12.9 29 35 41 a The concentration of the carbon sources in the growth medium was 0.4%. b Strain EWH80 is rel, and Q2 and Qs are, respectively, an rel+ and rel isogenic pair. The generation times for these strains are as follows: for EWH80-acetate, 140 min, glucose-SMM, 25 min; and for Q2 and Q3-acetate, 140 min, glucoseSMM, 30 min.

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in acetate and glucose-SMM, and the activities of the lysyl-, arginyl-, seryl-, and valyl-tRNA synthetases were assayed. In all four cases, the activities of these enzymes were higher in acetate medium in the rel strain (Table 8). The ratio of activity for each enzyme in these two media (glucose-SMM-acetate) was always smaller in the rel strain. DISCUSSION Several lines of evidence, presented in this manuscript, indicate that the enhanced activity of lysyl-tRNA synthetase in the SAM synthetase mutants is not due to a second mutation, which merely increases the synthetic rate ofthe enzyme. By two biochemical criteria, susceptibility to thermal inactivation and apparent sensitivity toward endogenous proteases, the lysyltRNA synthetase in the metK mutant RG62 is quite different from the corresponding synthetase in its isogenic parent when both strains are grown in minimal medium. In contrast, the properties of lysyl-tRNA synthetase from the wild-type strain grown in minimal medium plus 3 mM glycyl-ileucine are similar to that of the synthetase from RG62. From a genetic standpoint, there would be a low probability that the same double mutation would occur in three independently isolated spontaneous mutants, unless they were very closely linked. The experiment with the merodiploid RG317 (metK+/metK) shows that, under conditions where the metK+ allele is dominant (13), the lysyl-tRNA synthetase activity returns to normal levels. If a second mutation were involved it would have to be present in the region covered by the F-prime factor, KLF16. However, we have recently learned (J. C. Patte and E. Boy, personal communication) that the lysyltRNA synthetase structural gene has been tentatively mapped in the lac proA region of the chromosome, which is far from the metK region. The weight of all of this evidence makes it seem highly unlikely that a mutation other than that in SAM synthetase is responsible for the change in activity and properties of lysyltRNA synthetase in the metK mutants. At this time it is important to interpret the above results with caution. It is tempting to attribute the difference in the properties of lysyl-tRNA synthetase in RG62 and its isogenic parent to an alteration in the state of methylation (the synthetase in RG62 would be expected *to lack methylation). However, in the absence of direct evidence for methylation of this enzyme, the data would also be consistent for a role for SAM synthetase itself, or for methylation of a factor that could modify the properties of lysyl-tRNA synthetase. Two metabolites that

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could potentially fit the latter description are the polyamines, spermidine and spermine. These compounds require S-adenosylmethione for their synthesis (32) and have been shown to interact with tRNA (28, 32) and aminoacyltRNA synthetases (14, 20). However, addition of these polyamines (100 pmg/ml) to RG62 grown in minimal medium had no effect on lysyltRNA synthetase activity (unpublished data). It is not presently understood where the small molecules glycyl-L-leucine and Lalanine, which have been shown to alter the properties of lysyl-tRNA synthetase in vivo (4, 8), exert their effect. It would now appear that growing the cells in the presence of the peptide is tantamount to interfering with the cellular methylation process. If correct, this would create a number of potential sites of action for the peptide. We have found that the peptide does not inhibit SAM synthetase activity in vivo or in vitro (unpublished data). Alternatively, the peptide may accomplish the same end result as lack of methylation, but by a different mechanism. In addition to its effect on lysyl-tRNA synthetase, glycyl-ileucine can also influence the activity of seryl-, arginyl-, and valyl-tRNA synthetases. The latter two, however, could be stimulated by the peptide only in the enriched medium, whereas seryl-, like lysyl-tRNA synthetase, was influenced by the metabolite upon cell growth in all media. Likewise seryl-tRNA synthetase activity was weakly enhanced in the metK mutant, RG62. Although the metK mutants are reported to have only 3% of the normal SAM synthetase activity, they are only marginally deficient in their methylation capacity (7). Greene et al. (7) have shown that DNA methylation is normal in strain RG62. Cyclopropane-fatty acid synthesis, which requires S-adenosylmethione, the product of SAM synthetase, was somewhat deficient, however (7). These results suggest that lysyl-tRNA synthetase could be a poorer substrate for methylation than are other proteins, and this deficiency would be more readily apparent in a marginal situation. If a more defective SAM synthetase mutant could be isolated, it is quite possible that seryl-tRNA synthetase would display a more definitive response, and other proteins, which at present are not affected, would be. The data presented here is the first instance in which methylation has been shown to have a potential role in the control of aminoacyl-tRNA synthetase activity. The structure of glutamyltRNA synthetase can also apparently be influenced by mutations that map outside of the structural gene region (16, 21). In the past several years two modes of regula-

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tion of the aminoacyl-tRNA synthetases have been proposed (23, 27). The first model was patterned after the repressive control of amino acid biosynthesis and is thought to be governed by the degree of aminoacylation of tRNA species (37). According to this type of regulatory mechanism, a low in vivo level of aminoacylation of a tRNA should and can result in an increase in the level of its cognate synthetase (18). However, it has been clearly demonstrated that this form of control is not applicable to the influence of the metabolites such as glycyl-Lleucine on the lysyl-tRNA synthetase (11). Parker and Neidhardt subsequently proposed a model for the control of aminoacyl-tRNA synthetases called metabolic regulation (27). This has been followed by other publications on this mechanism of control (19, 23, 26), which ties the regulation of the synthetases into that of the translational apparatus. Although we have thought that there might be a relationship between this model and the effect of glycyl-Lleucine or L-alanine, the data presented in Tables 6 through 8 make it clear that these two forms of metabolic control act independently. Up to the present there has been little indication of what mechanism(s) operates in the metabolic control of the aminoacyl-tRNA synthetases. The highly phosphorylated nucleotide guanosine tetraphosphate (ppGpp) has been strongly implicated in the control of ribosomal RNA synthesis (29, 34, 35). It would therefore not be unexpected that the stringent control system would have some influence on the control of the aminoacyl-tRNA synthetases if the latter is coupled to the synthesis of other components of the translational apparatus such as ribosomal RNA. While we were working on this project, Blumenthal et al. (3) independently found by another approach that the stringent control system can influence the synthesis of the arginyl- and valyl-tRNA synthetases. Our data appear to corroborate those of Blumenthal et al. (3) in two respects: (i) the lysyl-tRNA synthetase appears to be less subject to the stringent control system than are the arginyl or valyl-tRNA synthetases, and (ii) stringent control alone does not appear to account for the metabolic regulation of the aminoacyl-tRNA

synthetases. ACKNOWLEDGMENTS We express our gratitude to those individuals, particularly R. Greene, who provided us with bacterial strains. We also thank the following for their support: The National Science Foundation (BMS74-19693); the National Institute of General Medical Sciences (Public Health Service grant 6 R01-GM21529-02); and the American Cancer Society (NP-2S).

J. BACTERIOL. LITERATURE CITED 1. Andrews, P. 1970. Estimation of molecular size and molecular weights of biological compounds by gel filtration, p. 1-63. In D. Glick (ed.), Methods of biochemical analysis, vol. 18. Academic Press Inc., New York. 2. Been, R. F., Jr., and I. W. Sizer. 1962. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 196:133140. 3. Blumenthal, R. M., P. G. Lemaux, F. C. Neidhardt, and P. P. Dennis. 1976. The effects of the rel A gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in E. coli B. Mol. Gen. Genet. 149:291-296. 4. Buklad, N. E., D. Sanborn, and I. N. Hirshfield. 1973. Particular influence of leucine peptides on lysyltransfer ribonucleic acid ligase formation in a mutant of Escherichia coli K-12. J. Bacteriol. 116:1477-1478. 5. Davis, B. D., and E. S Mingioli. 1960. Mutants ofEscherichia coli requiring methionine or vitamin B,2. J. Bacteriol. 60:17-28. 6. Dittgen, R. M., and R. Leberman. 1976. Multiple forms of lysyl-tRNA synthetase from Escherichia coli. Hoppe-Seyler's Z. Physiol. Chem. 357:643-551. 7. Greene, R. C., J. S. V. Hunter, and E. H. Coch. 1973. Properties of metK mutants of Escherichia coli K-12. J. Bacteriol. 115:57-67. 8. Hirshfleld, I. N., and N. E. Buklad. 1973. Effect of alanine, leucine, and fructose on lysyl-transfer ribonucleic acid ligase activity in a mutant ofEscherichia coli K-12. J. Bacteriol. 113:167-177. 9. Hirshfield, I. N., and F.-M. Yeh. 1976. An in vivo effect of the metabolites L-alanine and glycyl-L-leucine on the properties of lysyl-tRNA synthetase from Ewcherichia coli K-12. II. Kinetic studies. Biochim. Biophys. Acta 435:306-314. 10. Hirshfield, I. N., and P. C. Zamecnik. 1972. Thiosineresistant mutants of Escherichia coli K-12 with growth medium dependent lysyl-tRNA synthetase activity. I. Isolation and physiological characterization. Biochim. Biophys. Acta 259:330-343. 11. Hirshfield, I. N., F.-M. Yeh, and L. E. Sawyer. 1975. Metabolites influence control of lysine transfer ribonucleic acid synthetase formation in Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 72:1364-1367. 12. Hirshfield, I. N., F.-M. Yeh, and P. C. Zamecnik. 1976. An in vivo effect of the metabolites L-alanine and glycyl-L-leucine on the properties of lysyl-tRNA synthetase from Escherichia coli K-12. I. Influence on subunit composition and molecular weight distribution. Biochim. Biophys. Acta 435:290-305. 13. Hunter, J. S. V., R. C. Greene, and C.-H. Su. 1975. Genetic characterization of the metK locus in Ewcherichia coli K-12. J. Bacteriol. 122:1144-1152. 14. Iarashi, K., K. Matsuzaki, and Y. Takedo. 1971. Aminoacyl-transfer RNA formation. I. Absence of pyrophosphate-ATP exchange in aminoacyl-tRNA formation stimulated by polyamines. Biochim. Biophys. Acta 254:91-103. 15. Kornberg, A. 1966. Lactic dehydrogenase of muscle, p. 441-443. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 1. Academic Press Inc., New York. 16. Lapointe, J., and G. Delcuve. 1975. Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic subunit and in a regulator factor of the glutamyl-transfer ribonucleic acid synthetase. J. Bacteriol. 122:352-358. 17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

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18. McGinnis, E., and L. S. Williams. 1972. Regulation of histidyl-transfer ribonucleic acid synthetase formation in a histidyl-transfer ribonucleic acid synthetase mutant of Salmonella typhimurium. J. Bacteriol. 111:739-744. 19. McKeever, W. G., and F. C. Neidhardt. 1976. Growth rate modulation of four aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli. J. Bacteriol. 126:634445. 20. Matsuzaki, K., and Y. Takeda. 1973. Aminoacyl transfer RNA formation. III. Mechanism of aminoacylation stimulated by polyamines. Biochim. Biophys. Acta 308:339-351. 21. Murgola, E. J., and E. A. Adelberg. 1970. Mutants of Escherichia coli K-12 with an altered glutamyl-transfer ribonucleic acid synthetase. J. Bacteriol. 103:178183. 22. Neidhardt, F. C., P. L. Block, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. 119:736-747. 23. Neidhardt, F. C., J. Parker, and W. G. McKeever. 1975. Function and regulation of aminoacyl-tRNA synthetases in prokaryotic and eukaryotic cells. Annu. Rev. Microbiol. 29:215-250. 24. Novick, R. P., and W. K. Maas. 1961. Control by endogenously synthesized arginine of the formation of ornithine transcarbamylase in Escherichia coli. J. Bacteriol. 81:236-240. 25. Paik, W. I., and S. Kim. 1975. Protein methylation: chemical, enzymological and biological significance. Adv. Enzymol. 42:227-286. 26. Parker, J., M. Flashner, W. G. McKeever, and F. C. Neidhardt. 1974. Metabolic regulation of the arginyl and valyl-transfer ribonucleic acid synthetases in bacteria. J. Biol. Chem. 249:1044-1053. 27. Parker, J., and F. C. Neidhardt. 1972. Metabolic regu-

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