Vol. 123, No. 2
JOURNAL OF BACTERIOLOGY, Aug. 1975, p. 589-597 Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Role of Methionyl-Transfer Ribonucleic Acid in the Regulation of Methionyl-Transfer Ribonucleic Acid Synthetase of Escherichia coli K-12 D. CASSIOl de Laboratoire d'Enzymologie, Centre National la Recherche Scientifique, 91190 Gif-sur-Yvette, France Received for publication 22 April 1975
A decrease in the in vivo acylation level of methionine transfer ribonucleic acid (tRNAmet) induced by methioninyl adenylate led to a specific derepression of methionyl-transfer ribonucleic acid (tRNA) synthetase formation. This derepression required de novo protein synthesis and was reflected by overproduction of unaltered enzyme. Two different strains of Escherichia coli K-12 that have normal levels of methionyl-tRNA synthetase were examined and the derepression of methionyl-tRNA synthetase was observed in both. Moreover, for one of these strains, the relation between the level of methionyl-tRNA synthetase and deacylation level of tRNAmet was established; under the growth conditions used, when more than 25% of tRNAmet was deacylated, methionyl-tRNA synthetase formation was derepressed and the level of derepression became proportional to the amount of tRNAmet deacylated. Concomitantly, the enzyme was subject to specific inactivation as a consequence of which the true de novo rate of derepression of the formation of this enzyme was higher than that determined by measurements of enzyme activity. These studies were extended to strains AB311 and ed2, which had a constitutive enhanced level of methionyl-tRNA synthetase. In these strains no derepression of enzyme formation was observed on reducing the acylation level of tRNAmet by use of methioninyl adenylate.
In bacteria, the aminoacyl-transfer ribonucleic acid (tRNA) synthetases are not synthetized constitutively. The regulation of their synthesis by a process of repression apparently mediated by the corresponding amino acid has been established for at least 10 of the 20 aminoacyl-tRNA synthetases (for review, see reference 14). Moreover, in a few cases the aminoacyl-tRNA has been implicated in this regulation mechanism (4, 18). In a previous report we have shown that aminoalkyl adenylates, which are very specific potent inhibitors of aminoacyl-tRNA synthetases in vitro, can operate also in vivo since they inhibit growth of Escherichia coli K-12 (10). Recently we have established that the bacteriostatic power of L-methionyl adenylate is a consequence of its action on methionyl-tRNA synthetase (8). Thus, by using this product it becomes possible to modulate specifically and accurately the aminoacylation level of methionine tRNA (tRNAmet) in vivo. By this means the relation of growth rate to the fraction of tRNAmet aminoac-
ylated in vivo has been determined for E. coli K-12 strain HfrH (8). In the present report we have investigated the consequences on the synthesis of methionyl-tRNA synthetase of reducing the level of acylation of tRNAmet by use of L-methioninyl adenylate. The study was carried out on several strains of E. coli K-12, in particular on those that have an enhanced level of methionyl-tRNA synthetase.
1Present address: Centre de G6netique Moleculaire, Centre National de la Recherche Scientifique, 91190 Gif-surYvette, France.
589
MATERIALS AND METHODS Bacterial strains. Seven strains of E. coli K-12 were examined in this study. The strain used as reference was HfrH (thi-1) (5). Strain AB311 (thr-1, leu-6, thi-1, str-8) and the P2-mediated eductant ed2 derived from this strain were kindly provided by B. Rotman. This eductant had been isolated as a histidine-requiring strain and corresponds to strain QE76 described by Sunshine and Kelly (20). Strains Y1OF-, W208F-, W208,SRF-, and AB284 F+, which are parental strains of AB311 (5), were obtained from B. Bachmann. Strain AB284 F+ was cured of the sex factor F by growing overnight in the presence of 50 ,g of acridine orange per ml as described by Miller (19). After this treatment, the fraction of cured cells was estimated at 100% on the basis of resistance to male-specific phage MS2 (19). Three independent cured colonies were used in the experiment described.
590
J. BACTERIOL.
CASSIO
Media and methods of cultivation. Strains were grown at 37 C in minimal medium 63 (13) containing thiamine at 0.5 mg/ml with 0.4% glucose as the carbon source supplemented with required growth factors at 0.5 mM. Growth was monitored by absorbance at 420 nm. Overnight cultures in exponential phase were diluted at approximately 5 x 107 cells/ml (absorbance = 0.13) with fresh medium, and on reaching an absorbance of 0.35 methioninyl adenylate at the appropriate concentration was added. Growth was allowed to proceed at the new growth rate imposed by methioninyl adenylate and, unless otherwise indicated, cells were harvested in exponential phase (absorbance = 2.8). Preparation of cell extracts. Approximately 1010 to 2 x 1010 cells were suspended in 7 ml of 20 mM potassium phosphate buffer at pH 7.6 containing 5 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 x 2 liters of the same buffer. The protein content was determined by the method of Lowry et al. (16), using bovine serum albumin as the standard. Measurements of the activities of the aminoacyltRNA synthetases. These activities were measured by the rate of aminoacyl-tRNA formation as described earlier (15) except that the pH was 7.8. One unit of enzyme activity is defined as the amount producing aminoacylation of 1 nmol of tRNA at 30 C in 10 min. Material. L-["4C]methionine and L-["4C]valine (50 mCi/mmol) were obtained from the Commissariat a l'Energie Atomique (Saclay, France). L-Methioninyl adenylate synthetized as described previously (4) was kindly provided by M. Robert-Gero. The antiserum used was directed against purified methionyl-tRNA synthetase from E. coli K-12 strain EM20031 as reported earlier (12). Unfractionated tRNA was prepared from the same strain according to Zubay (22).
RESULTS Methionyl-tRNA synthetase formation in E. coli strain HfrH grown in the presence of methioninyl adenylate. In the first series of experiments, strain HfrH was grown during three generations in medium supplemented with different concentrations of methioninyl adenylate and the level of methionyl-tRNA synthetase was measured. In addition, the level of valyl-tRNA synthetase was determined as a control. Increasing the concentration of the inhibitor in the growth medium resulted in a decrease of growth rate as earlier described (8) and in a concomitant increase of the specific activity of methionyl-tRNA synthetase (Fig. 1); under these growth conditions the level of valyl-tRNA synthetase activity remained unchanged. This augmentation in methionyl-tRNA syn-
.o
.
d
z z
I
I
3
o
iz lx cr
100
_--
..z O
M.
50
0.3 0.2 0.1 methioninyl adenylate (mM) FIG. 1. Effect of methioninyl adenylate on growth rate and on levels of methionyl- and valyl-tRNA synthetases of strain HfrH. Strain HfrH was grown at 37 C for three generations in the presence of different concentrations of methioninyl adenylate as described in the text. Growth rate of 100% corresponds to a generation time of 63 min. Level of 100% in enzyme activities corresponds to a specific activity of 39 and 117 U/mg for methionyl- and valyl-tRNA synthetases,
respectively.
thetase activity could result from the production of' a more active enzyme or from an increase in the synthesis of' unaltered enzyme. To distinguish between these two alternatives, the neutralization curves of methionyl-tRNA svnthetase activity f'rom cells grown in the presence and absence of methioninyl adenylate by antibodies directed against purified enzyme were determined. The activity of methionyl-tRNA synthetase in a crude extract of strain HfrH grown in the presence of 0.2 mM L-methioninyl adenylate was 2.2 times greater than that of the same strain grown in the absence of this inhibitor and required 2.2 times more antibodies for neutralization (Fig. 2). In addition, the molecular weight of methionyl-tRNA synthetase from extracts of HfrH grown in the absence and presence of methioninyl adenylate was investigated by f'iltration through Sephadex G-200. In each case, all of the enzymatic activity was eluted in one single component, the elution volume of which corresponded to a molecular
VOL. 123, 1975
METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. II.
2000 1-
E
L-
q6-
.
E
0-
1o
0
10 concentration
20 or
30
40
antiserum (lL. protein /m
)
FIG. 2. Neutralization of methionyl-tRNA synthetase activity in crude extracts of strain HfrH grown in the absence and presence of methioninyl adenylate. Strain HfrH was grown in the absence and presence of 0.2 mM methioninyl adenylate during three generations as described in the text. Crude extracts from such cultures (each at a protein concentration of 90 yg/ml in 20 mMpotassium phosphate buffer at pH 7.6 containing 200 gg of bovine serum albumin per 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 after the appropriate dilution methionyl-tRNA formation was measured; methionyl-tRNA synthetase activity of cells grown in the absence (0) and presence of 0.2 mM methionyl adenylate (0).
weight of 175,000. These results suggest that the increased level of methionyl-tRNA synthetase in cells grown in the presence of methioninyl adenylate is due to an overproduction of unaltered enzyme. The possibility was considered that this specific increase of methionyl-tRNA synthetase level observed after growth in the presence of methioninyl adenylate was a consequence of the reduction of growth rate. This hypothesis was tested and discarded by use of chloramphenicol. Indeed, as reported earlier (8), in the presence of low concentrations of this antibiotic, the growth rate of E. coli was reduced but stayed constant, as in the presence of aminoalkyl adenylates. When cells were cultivated for three generations in the presence of 3 ug of chloramphenicol per ml, which reduced the growth rate by half, the specific activities of methionyl- and valyl-tRNA synthetases remained unaltered.
591
Archibold and Williams (3) have reported that methionine-restricted growth caused derepression of' the rate of synthesis of methionyl-tRNA synthetase in an auxotrophic strain of E. coli K-12. However, they f'urther observed that the level of' this derepression was not constant: during the f'irst minutes af'ter the transfer from unrestricted to limiting methionine medium, a 48-f'old increase in the specif'ic activitv of the enzyme occurred. In contrast, in cells grown during one generation in methionine-limiting medium there was onlv a 2.5-t'old increase in the rate of synthesis of' methionyl-tRNA svnthetase as determined by density labeling. In view of' these data, the variation of' methionyl-tRNA synthetase level was measured throughout growth in the presence of methioninyl adenylate. The addition of 0.17 mM methioninyl adenylate to exponentially growing cells of strain HfrH induced a progressive increase of the. specific activity of methionyltRNA synthetase (Fig. 3). After 1 h of growth in the presence of the inhibitor, the level of this enzyme became constant and reached 1.8-fold that of the cells grown in the absence of methioninyl adenylate. Moreover, when chloramphenicol (300 Hg/ml) was added at the same time as methioninyl adenylate, no increase in activity of methioninyl-tRNA synthetase was observed, indicating that the augmentation in enzyme level that occurs during growth in the presence of methioninyl adenylate requires de novo protein synthesis. Under these conditions, the level of valyl-tRNA synthetase remained unchanged whereas the level of methionyl-tRNA synthetase decreased progressively. This decrease is attributed to a specific inactivation of the methionyl-tRNA synthetase as discussed later (see Fig. 7). In Fig. 4 a differential plot of the preceding data is presented. It appears that the progressive increase in methionyl-tRNA synthetase level observed after addition of' 0.17 mM methioninyl adenylate to growing cells of strain HfrH corresponds to a fivefold derepression in the rate of formation of this enzyme. Moreover and as expected, the presence of' the inhibitor in the growth medium had no effect on the rate of formation of valyl-tRNA synthetase (Fig. 4). In a previous report (8) we have shown that in cells of strain HfrH grown exponentially in minimal medium, tRNAmet is aminoacylated at 100% and that on addition of methioninyl adenylate to the growth medium there is a specific deacylation of this tRNA. Moreover, for this strain, the relation of growth rate to the aminoacylation level of tRNAmet has been established (8). In Fig. 5 the level of methionyl-tRNA syn-
592
J. BACTERIOL.
CASSIO
150
Effect of methioninyl adenylate on methionyl-tRNA synthetase formation in E. coli K-12 strains that have an enhanced level of this enzyme. We have previously shown that strain AB311 is characterized by a constitutive threefold increased level of methionyl-tRNA
~
200
-
~~~~~~~~~~~~10
63
-.
100
Z8 *~50-6E
z
0
1
2
3
4
time (hr) FIG. 3. Variation of methionyl-tRNA synthetase level in strain HfrH during growth in the presence of 0.17 mM methioninyl adenylate. A culture of exponentially growing cells of strain HfrH (1.5 x 10' cells/ml) was divided into three flasks, one that served as control, one that received methioninyl adenylate (0.17 mM), and another that rqceived methioninyl adenylate (0.17 mM) and chloramphenicol (300 gg/ml). At different times after the addition of these products, 7j samples were removed and methionyl- and valyltRNA synthetase activities were determined for the control culture (0), for the culture grown in the presence of methioninyl adenylate (0), and for the , culture to which methioninyl adenylate and chloram' phenicol were added (A). In the three cultures exam' ined, the levels of valyl-tRNA synthetase were the same. The 100% level of methionyl-tRNA synthetase z corresponds to a specific activity of 37 U/mg. Growth i rates in the absence and presence of methioninyl adenylate correspond to a generation time of 60 and 84 min, respectively.
thetase measured in cells grown during three generations in the presence of different concentrations of methioninyl adenylate is presented in terms of deacylation level of tRNAmet determined growth mined in these cells under the ame growth s same conditions. It appears that, when more than 25% of tRNAmet is deacylated, the formation of methionyl-tRNA synthetase is derepressed and the level of derepression becomes proportional to the amount of tRNAmet deacylated.
cells.under
2
l
l-1
10 _
5
5
0
0.05 0.10 0.15 bacterial protein(mg/ml)
FIG. 4.rate Effect of methioninyl adenylate on the differential of formation of methionyl-tRNA synthe-
tase. Experimental conditions are as described in Fig. 3. The results are expressed as enzyme units per
milliliter of culture as a function of total protein per milliliter of culture of cells grown in the absence (0) and presence (0) of methioninyl adenylate.
VOL. 123, 1975
METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. II.
pedigree that has a threefold higher level of methionyl-tRNA synthetase than usual. Moreover, this increased level is not related to the presence of the sex factor, since strain AB284 F-, isolated from AB284 F+ after curing by acridine orange, still has three times more methionyl-tRNA synthetase. From these results it appears that in the strain isolated as AB284 F+ the event that was responsible for a threefold increase in the level of methionyl-tRNA synthetase occurred fortuitously and independently of the sex factor transfer. On the other hand, we have recently examined strains termed eductants that carry P2-mediated deletions in the proximity of the structural gene of methionyl-tRNA synthetase (9). It was found that in two eductants isolated from strain
0
SD
z
ca a#
m
593
TABLE 1. Level of methionyl- and valyl-tRNA synthetases in different strains related to strain AB311
.
4.
Strains of the genealogy of Hfr AB311a
Methionyl-tRNA synthetase (relative sp act)b
Valyl-tRNA synthetase (relative sp act)"
1.0
1.0
1.0
1.0
1.1
1.1
3.0
1.0
3.1
0.9
2.9 3.1 3.2
0.9 0.9 1.0
Y1OFthr-1 leu-6
thi-1 supE44 4uv W208Fthr-1 leu-6 c thi-1
sup-49
0 25 50 75 100 level of deacylation of tRNA met FIG. 5. Level of methionyl-tRNA synthetase as a function of the deacylation level of tRNAMe in strain HfrH grown in the presence of different concentrations of methioninyl adenylate. We have previously measured the level of acylation of tRNA-" in strain HfrH grown during three generations in the presence of different concentrations of methioninyl adenylate and the relation between growth rate and level of acylation of tRNA-"e was determined (5). Based on this relation, the level of methionyl-tRNA synthetase in strain HfrH grown in precisely the same conditions is presented in terms of the deacylation level of tRNAmet.
lacZ4
W208,SRF$cI str-8 Fl from o wild type AB284 F+ {C} str-8
4.uv
AB31lHfr
{Ci str-8 X defective
Poll AB284F -c
synthetase (7) that is probably due to an increase in net synthesis of this enzyme (12). The molecular event or the selecting agent responsible for this increase is unknown. However, the genealogical step leading to this increase was determined. As reported in Table 1, strain AB248 F+ is the first strain in the AB311
According to Bachmann (5). h Relative to HfrH c Strain AB284 F+ was cured of the sex factor F as described in the text. The experimental results refer to three independent cured colonies. a
594
J. BACTERIOL.
CASSIO
AB311 (ed2 and edlO) the level of this enzyme reached twice the level found in the parental strain. In addition, we have shown that this higher level is not due to the production of an altered enzyme but to an increase in the net synthesis of the enzyme (9). It was of some interest to determine whether in strains such as AB311 or ed2. which have an enhanced level of methionyl-tRNA synthetase, the formation of the enzyme is repressed by methionyl-tRNA. Theref'ore, these two strains were grown in the presence of methioninyl adenylate, and the methionyl- and valyl-tRNA synthetase activities were measured. For control purposes, one of the parental strains, W208F-, which has a normal level of methionyl-tRNA synthetase, was also examined. For strain W208F-, a decrease in the cellular level of' methionyl-tRNA induced by methioninyl adenylate led to a derepression in the level of methionyl-tRNA synthetase as previously reported in the case of strain HfrH (Fig. 6). In contrast, under the same conditions there was no derepression of' the enzyme in strains AB311 and ed2. For these two strains it was observed that the more the growth was inhibited by methioninyl adenylate, the lower was the activity of methionyl-tRNA synthetase, whereas the activity of valyl-tRNA synthetase remained unchanged. This decrease in methionyl-tRNA synthetase level probably reflects inactivation of the enzyme under the growth conditions used. To get an insight into the nature of this presumed inactivation, the methionyl-tRNA synthetase of strain AB311 grown for three generations in the presence of 0.65 mM methioninyl adenylate was characterized. Under these conditions, the level of methionyl-tRNA synthetase was 40% of that found in the absence of the inhibitor. The apparent molecular weight antigenic properties, and catalytic parameters of the enzyme were determined. It was found that all of the methionyl-tRNA synthetase activity was catalyzed by unaltered enzyme. Moreover, no inactive material reacting with antibodies directed against normal enzyme was detected.
DISCUSSION A repression-derepression mechanism f'or the regulation of enzyme synthesis has been shown f'or a number of' aminoacyl-tRNA svnthetases (14). Culture conditions limiting the supply of' an amino acid will often specif'ically derepress the synthesis of' the cognate aminoacyl-tRNA synthetase. However, it is not always easy to observe the increase rate of' enzyme synthesis because of inactivation under some conditions
of' amino acid starvation (21). In a few cases, the aminoacyl-tRNA has been implicated in the repression of the cognate aminoacyl-tRNA synthetase. Thus, McGinnis and Williams have examined the role of tRNAhis in repression of' histidyl-tRNA synthetase formation in two strains of Salmonella typhimurium, a wildtype strain, and a tRNAhlS mutant (hisR) possessing only 52% of' the histidine acceptor activity of the wild-type strain (18). In both strains, histidyl-tRNA synthetase formation was derepressed during histidine-restricted growth. However, restoration of' histidine to the derepressed wild-type culture caused an immediate cessation of synthesis of' the enzyme, whereas the hisR mutant continued to synthetize the histidvl-tRNA synthetase at the derepressed rate despite the presence of' histidine. More recently, the same authors have investigated the involvement of histidyl-tRNA synthetase in regulating the expression ot' its own structural gene ( 17), and their results support the idea that histidyl-tRNA is the corepressor (or repressor) for histidyl-tRNA synthetase formation. In addition, in the case of methionyl-tRNA synthetase of' E. coli and S. typhimurium, it appears f'rom the examination of mutants of' this enzyme (4) that synthetase alteration(s) results in a derepression of synthesis of the enzyme due to a reduction in the acylation level of tRNAmet. However, the study of' the role of' aminoacyl-tRNA's in the regulation of aminoacyl-tRNA synthetases has proven difficult and laborious since it requires obtaining and characterizing mutants with altered synthetases or with altered tRNA's. In a previous report (9) we postulated that aminoalkyl adenvlates, considering their specif'ic action in vivo, should proxve usef'ul in investigations on the possible involvement of aminoacyl-tRNA's in regulation processes. This assertion is supported by the results presented in the present report. Indeed, it is shown that in E. coli K-12 a decrease in intracellular concentration of methionvl-tRNA induced by methioninyl adenylate leads to a specific derepression of the synthesis of methionyl-tRNA synthetase. Moreover, in the case of' strain HfrH, the relation between the level of methionyl-tRNA svnthetase and deacylation level of tRNAmet was determined: in cells grown in minimal medium, more than 25%, of tRNA-et is deacylated, methionyl-tRNA synthetase formation is derepressed and the level ot' derepression becomes proportional to the amount of tRNAmet deacylated. These results strongly support the hvpothesis that methionvl-tRNA is implicated
VOL. 123, 1975
300
METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. II.
-
JOURNAL OF BACTERIOLOGY, Aug. 1975, p. 589-597 Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Role of Methionyl-Transfer Ribonucleic Acid in the Regulation of Methionyl-Transfer Ribonucleic Acid Synthetase of Escherichia coli K-12 D. CASSIOl de Laboratoire d'Enzymologie, Centre National la Recherche Scientifique, 91190 Gif-sur-Yvette, France Received for publication 22 April 1975
A decrease in the in vivo acylation level of methionine transfer ribonucleic acid (tRNAmet) induced by methioninyl adenylate led to a specific derepression of methionyl-transfer ribonucleic acid (tRNA) synthetase formation. This derepression required de novo protein synthesis and was reflected by overproduction of unaltered enzyme. Two different strains of Escherichia coli K-12 that have normal levels of methionyl-tRNA synthetase were examined and the derepression of methionyl-tRNA synthetase was observed in both. Moreover, for one of these strains, the relation between the level of methionyl-tRNA synthetase and deacylation level of tRNAmet was established; under the growth conditions used, when more than 25% of tRNAmet was deacylated, methionyl-tRNA synthetase formation was derepressed and the level of derepression became proportional to the amount of tRNAmet deacylated. Concomitantly, the enzyme was subject to specific inactivation as a consequence of which the true de novo rate of derepression of the formation of this enzyme was higher than that determined by measurements of enzyme activity. These studies were extended to strains AB311 and ed2, which had a constitutive enhanced level of methionyl-tRNA synthetase. In these strains no derepression of enzyme formation was observed on reducing the acylation level of tRNAmet by use of methioninyl adenylate.
In bacteria, the aminoacyl-transfer ribonucleic acid (tRNA) synthetases are not synthetized constitutively. The regulation of their synthesis by a process of repression apparently mediated by the corresponding amino acid has been established for at least 10 of the 20 aminoacyl-tRNA synthetases (for review, see reference 14). Moreover, in a few cases the aminoacyl-tRNA has been implicated in this regulation mechanism (4, 18). In a previous report we have shown that aminoalkyl adenylates, which are very specific potent inhibitors of aminoacyl-tRNA synthetases in vitro, can operate also in vivo since they inhibit growth of Escherichia coli K-12 (10). Recently we have established that the bacteriostatic power of L-methionyl adenylate is a consequence of its action on methionyl-tRNA synthetase (8). Thus, by using this product it becomes possible to modulate specifically and accurately the aminoacylation level of methionine tRNA (tRNAmet) in vivo. By this means the relation of growth rate to the fraction of tRNAmet aminoac-
ylated in vivo has been determined for E. coli K-12 strain HfrH (8). In the present report we have investigated the consequences on the synthesis of methionyl-tRNA synthetase of reducing the level of acylation of tRNAmet by use of L-methioninyl adenylate. The study was carried out on several strains of E. coli K-12, in particular on those that have an enhanced level of methionyl-tRNA synthetase.
1Present address: Centre de G6netique Moleculaire, Centre National de la Recherche Scientifique, 91190 Gif-surYvette, France.
589
MATERIALS AND METHODS Bacterial strains. Seven strains of E. coli K-12 were examined in this study. The strain used as reference was HfrH (thi-1) (5). Strain AB311 (thr-1, leu-6, thi-1, str-8) and the P2-mediated eductant ed2 derived from this strain were kindly provided by B. Rotman. This eductant had been isolated as a histidine-requiring strain and corresponds to strain QE76 described by Sunshine and Kelly (20). Strains Y1OF-, W208F-, W208,SRF-, and AB284 F+, which are parental strains of AB311 (5), were obtained from B. Bachmann. Strain AB284 F+ was cured of the sex factor F by growing overnight in the presence of 50 ,g of acridine orange per ml as described by Miller (19). After this treatment, the fraction of cured cells was estimated at 100% on the basis of resistance to male-specific phage MS2 (19). Three independent cured colonies were used in the experiment described.
590
J. BACTERIOL.
CASSIO
Media and methods of cultivation. Strains were grown at 37 C in minimal medium 63 (13) containing thiamine at 0.5 mg/ml with 0.4% glucose as the carbon source supplemented with required growth factors at 0.5 mM. Growth was monitored by absorbance at 420 nm. Overnight cultures in exponential phase were diluted at approximately 5 x 107 cells/ml (absorbance = 0.13) with fresh medium, and on reaching an absorbance of 0.35 methioninyl adenylate at the appropriate concentration was added. Growth was allowed to proceed at the new growth rate imposed by methioninyl adenylate and, unless otherwise indicated, cells were harvested in exponential phase (absorbance = 2.8). Preparation of cell extracts. Approximately 1010 to 2 x 1010 cells were suspended in 7 ml of 20 mM potassium phosphate buffer at pH 7.6 containing 5 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 x 2 liters of the same buffer. The protein content was determined by the method of Lowry et al. (16), using bovine serum albumin as the standard. Measurements of the activities of the aminoacyltRNA synthetases. These activities were measured by the rate of aminoacyl-tRNA formation as described earlier (15) except that the pH was 7.8. One unit of enzyme activity is defined as the amount producing aminoacylation of 1 nmol of tRNA at 30 C in 10 min. Material. L-["4C]methionine and L-["4C]valine (50 mCi/mmol) were obtained from the Commissariat a l'Energie Atomique (Saclay, France). L-Methioninyl adenylate synthetized as described previously (4) was kindly provided by M. Robert-Gero. The antiserum used was directed against purified methionyl-tRNA synthetase from E. coli K-12 strain EM20031 as reported earlier (12). Unfractionated tRNA was prepared from the same strain according to Zubay (22).
RESULTS Methionyl-tRNA synthetase formation in E. coli strain HfrH grown in the presence of methioninyl adenylate. In the first series of experiments, strain HfrH was grown during three generations in medium supplemented with different concentrations of methioninyl adenylate and the level of methionyl-tRNA synthetase was measured. In addition, the level of valyl-tRNA synthetase was determined as a control. Increasing the concentration of the inhibitor in the growth medium resulted in a decrease of growth rate as earlier described (8) and in a concomitant increase of the specific activity of methionyl-tRNA synthetase (Fig. 1); under these growth conditions the level of valyl-tRNA synthetase activity remained unchanged. This augmentation in methionyl-tRNA syn-
.o
.
d
z z
I
I
3
o
iz lx cr
100
_--
..z O
M.
50
0.3 0.2 0.1 methioninyl adenylate (mM) FIG. 1. Effect of methioninyl adenylate on growth rate and on levels of methionyl- and valyl-tRNA synthetases of strain HfrH. Strain HfrH was grown at 37 C for three generations in the presence of different concentrations of methioninyl adenylate as described in the text. Growth rate of 100% corresponds to a generation time of 63 min. Level of 100% in enzyme activities corresponds to a specific activity of 39 and 117 U/mg for methionyl- and valyl-tRNA synthetases,
respectively.
thetase activity could result from the production of' a more active enzyme or from an increase in the synthesis of' unaltered enzyme. To distinguish between these two alternatives, the neutralization curves of methionyl-tRNA svnthetase activity f'rom cells grown in the presence and absence of methioninyl adenylate by antibodies directed against purified enzyme were determined. The activity of methionyl-tRNA synthetase in a crude extract of strain HfrH grown in the presence of 0.2 mM L-methioninyl adenylate was 2.2 times greater than that of the same strain grown in the absence of this inhibitor and required 2.2 times more antibodies for neutralization (Fig. 2). In addition, the molecular weight of methionyl-tRNA synthetase from extracts of HfrH grown in the absence and presence of methioninyl adenylate was investigated by f'iltration through Sephadex G-200. In each case, all of the enzymatic activity was eluted in one single component, the elution volume of which corresponded to a molecular
VOL. 123, 1975
METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. II.
2000 1-
E
L-
q6-
.
E
0-
1o
0
10 concentration
20 or
30
40
antiserum (lL. protein /m
)
FIG. 2. Neutralization of methionyl-tRNA synthetase activity in crude extracts of strain HfrH grown in the absence and presence of methioninyl adenylate. Strain HfrH was grown in the absence and presence of 0.2 mM methioninyl adenylate during three generations as described in the text. Crude extracts from such cultures (each at a protein concentration of 90 yg/ml in 20 mMpotassium phosphate buffer at pH 7.6 containing 200 gg of bovine serum albumin per 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 after the appropriate dilution methionyl-tRNA formation was measured; methionyl-tRNA synthetase activity of cells grown in the absence (0) and presence of 0.2 mM methionyl adenylate (0).
weight of 175,000. These results suggest that the increased level of methionyl-tRNA synthetase in cells grown in the presence of methioninyl adenylate is due to an overproduction of unaltered enzyme. The possibility was considered that this specific increase of methionyl-tRNA synthetase level observed after growth in the presence of methioninyl adenylate was a consequence of the reduction of growth rate. This hypothesis was tested and discarded by use of chloramphenicol. Indeed, as reported earlier (8), in the presence of low concentrations of this antibiotic, the growth rate of E. coli was reduced but stayed constant, as in the presence of aminoalkyl adenylates. When cells were cultivated for three generations in the presence of 3 ug of chloramphenicol per ml, which reduced the growth rate by half, the specific activities of methionyl- and valyl-tRNA synthetases remained unaltered.
591
Archibold and Williams (3) have reported that methionine-restricted growth caused derepression of' the rate of synthesis of methionyl-tRNA synthetase in an auxotrophic strain of E. coli K-12. However, they f'urther observed that the level of' this derepression was not constant: during the f'irst minutes af'ter the transfer from unrestricted to limiting methionine medium, a 48-f'old increase in the specif'ic activitv of the enzyme occurred. In contrast, in cells grown during one generation in methionine-limiting medium there was onlv a 2.5-t'old increase in the rate of synthesis of' methionyl-tRNA svnthetase as determined by density labeling. In view of' these data, the variation of' methionyl-tRNA synthetase level was measured throughout growth in the presence of methioninyl adenylate. The addition of 0.17 mM methioninyl adenylate to exponentially growing cells of strain HfrH induced a progressive increase of the. specific activity of methionyltRNA synthetase (Fig. 3). After 1 h of growth in the presence of the inhibitor, the level of this enzyme became constant and reached 1.8-fold that of the cells grown in the absence of methioninyl adenylate. Moreover, when chloramphenicol (300 Hg/ml) was added at the same time as methioninyl adenylate, no increase in activity of methioninyl-tRNA synthetase was observed, indicating that the augmentation in enzyme level that occurs during growth in the presence of methioninyl adenylate requires de novo protein synthesis. Under these conditions, the level of valyl-tRNA synthetase remained unchanged whereas the level of methionyl-tRNA synthetase decreased progressively. This decrease is attributed to a specific inactivation of the methionyl-tRNA synthetase as discussed later (see Fig. 7). In Fig. 4 a differential plot of the preceding data is presented. It appears that the progressive increase in methionyl-tRNA synthetase level observed after addition of' 0.17 mM methioninyl adenylate to growing cells of strain HfrH corresponds to a fivefold derepression in the rate of formation of this enzyme. Moreover and as expected, the presence of' the inhibitor in the growth medium had no effect on the rate of formation of valyl-tRNA synthetase (Fig. 4). In a previous report (8) we have shown that in cells of strain HfrH grown exponentially in minimal medium, tRNAmet is aminoacylated at 100% and that on addition of methioninyl adenylate to the growth medium there is a specific deacylation of this tRNA. Moreover, for this strain, the relation of growth rate to the aminoacylation level of tRNAmet has been established (8). In Fig. 5 the level of methionyl-tRNA syn-
592
J. BACTERIOL.
CASSIO
150
Effect of methioninyl adenylate on methionyl-tRNA synthetase formation in E. coli K-12 strains that have an enhanced level of this enzyme. We have previously shown that strain AB311 is characterized by a constitutive threefold increased level of methionyl-tRNA
~
200
-
~~~~~~~~~~~~10
63
-.
100
Z8 *~50-6E
z
0
1
2
3
4
time (hr) FIG. 3. Variation of methionyl-tRNA synthetase level in strain HfrH during growth in the presence of 0.17 mM methioninyl adenylate. A culture of exponentially growing cells of strain HfrH (1.5 x 10' cells/ml) was divided into three flasks, one that served as control, one that received methioninyl adenylate (0.17 mM), and another that rqceived methioninyl adenylate (0.17 mM) and chloramphenicol (300 gg/ml). At different times after the addition of these products, 7j samples were removed and methionyl- and valyltRNA synthetase activities were determined for the control culture (0), for the culture grown in the presence of methioninyl adenylate (0), and for the , culture to which methioninyl adenylate and chloram' phenicol were added (A). In the three cultures exam' ined, the levels of valyl-tRNA synthetase were the same. The 100% level of methionyl-tRNA synthetase z corresponds to a specific activity of 37 U/mg. Growth i rates in the absence and presence of methioninyl adenylate correspond to a generation time of 60 and 84 min, respectively.
thetase measured in cells grown during three generations in the presence of different concentrations of methioninyl adenylate is presented in terms of deacylation level of tRNAmet determined growth mined in these cells under the ame growth s same conditions. It appears that, when more than 25% of tRNAmet is deacylated, the formation of methionyl-tRNA synthetase is derepressed and the level of derepression becomes proportional to the amount of tRNAmet deacylated.
cells.under
2
l
l-1
10 _
5
5
0
0.05 0.10 0.15 bacterial protein(mg/ml)
FIG. 4.rate Effect of methioninyl adenylate on the differential of formation of methionyl-tRNA synthe-
tase. Experimental conditions are as described in Fig. 3. The results are expressed as enzyme units per
milliliter of culture as a function of total protein per milliliter of culture of cells grown in the absence (0) and presence (0) of methioninyl adenylate.
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METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. II.
pedigree that has a threefold higher level of methionyl-tRNA synthetase than usual. Moreover, this increased level is not related to the presence of the sex factor, since strain AB284 F-, isolated from AB284 F+ after curing by acridine orange, still has three times more methionyl-tRNA synthetase. From these results it appears that in the strain isolated as AB284 F+ the event that was responsible for a threefold increase in the level of methionyl-tRNA synthetase occurred fortuitously and independently of the sex factor transfer. On the other hand, we have recently examined strains termed eductants that carry P2-mediated deletions in the proximity of the structural gene of methionyl-tRNA synthetase (9). It was found that in two eductants isolated from strain
0
SD
z
ca a#
m
593
TABLE 1. Level of methionyl- and valyl-tRNA synthetases in different strains related to strain AB311
.
4.
Strains of the genealogy of Hfr AB311a
Methionyl-tRNA synthetase (relative sp act)b
Valyl-tRNA synthetase (relative sp act)"
1.0
1.0
1.0
1.0
1.1
1.1
3.0
1.0
3.1
0.9
2.9 3.1 3.2
0.9 0.9 1.0
Y1OFthr-1 leu-6
thi-1 supE44 4uv W208Fthr-1 leu-6 c thi-1
sup-49
0 25 50 75 100 level of deacylation of tRNA met FIG. 5. Level of methionyl-tRNA synthetase as a function of the deacylation level of tRNAMe in strain HfrH grown in the presence of different concentrations of methioninyl adenylate. We have previously measured the level of acylation of tRNA-" in strain HfrH grown during three generations in the presence of different concentrations of methioninyl adenylate and the relation between growth rate and level of acylation of tRNA-"e was determined (5). Based on this relation, the level of methionyl-tRNA synthetase in strain HfrH grown in precisely the same conditions is presented in terms of the deacylation level of tRNAmet.
lacZ4
W208,SRF$cI str-8 Fl from o wild type AB284 F+ {C} str-8
4.uv
AB31lHfr
{Ci str-8 X defective
Poll AB284F -c
synthetase (7) that is probably due to an increase in net synthesis of this enzyme (12). The molecular event or the selecting agent responsible for this increase is unknown. However, the genealogical step leading to this increase was determined. As reported in Table 1, strain AB248 F+ is the first strain in the AB311
According to Bachmann (5). h Relative to HfrH c Strain AB284 F+ was cured of the sex factor F as described in the text. The experimental results refer to three independent cured colonies. a
594
J. BACTERIOL.
CASSIO
AB311 (ed2 and edlO) the level of this enzyme reached twice the level found in the parental strain. In addition, we have shown that this higher level is not due to the production of an altered enzyme but to an increase in the net synthesis of the enzyme (9). It was of some interest to determine whether in strains such as AB311 or ed2. which have an enhanced level of methionyl-tRNA synthetase, the formation of the enzyme is repressed by methionyl-tRNA. Theref'ore, these two strains were grown in the presence of methioninyl adenylate, and the methionyl- and valyl-tRNA synthetase activities were measured. For control purposes, one of the parental strains, W208F-, which has a normal level of methionyl-tRNA synthetase, was also examined. For strain W208F-, a decrease in the cellular level of' methionyl-tRNA induced by methioninyl adenylate led to a derepression in the level of methionyl-tRNA synthetase as previously reported in the case of strain HfrH (Fig. 6). In contrast, under the same conditions there was no derepression of' the enzyme in strains AB311 and ed2. For these two strains it was observed that the more the growth was inhibited by methioninyl adenylate, the lower was the activity of methionyl-tRNA synthetase, whereas the activity of valyl-tRNA synthetase remained unchanged. This decrease in methionyl-tRNA synthetase level probably reflects inactivation of the enzyme under the growth conditions used. To get an insight into the nature of this presumed inactivation, the methionyl-tRNA synthetase of strain AB311 grown for three generations in the presence of 0.65 mM methioninyl adenylate was characterized. Under these conditions, the level of methionyl-tRNA synthetase was 40% of that found in the absence of the inhibitor. The apparent molecular weight antigenic properties, and catalytic parameters of the enzyme were determined. It was found that all of the methionyl-tRNA synthetase activity was catalyzed by unaltered enzyme. Moreover, no inactive material reacting with antibodies directed against normal enzyme was detected.
DISCUSSION A repression-derepression mechanism f'or the regulation of enzyme synthesis has been shown f'or a number of' aminoacyl-tRNA svnthetases (14). Culture conditions limiting the supply of' an amino acid will often specif'ically derepress the synthesis of' the cognate aminoacyl-tRNA synthetase. However, it is not always easy to observe the increase rate of' enzyme synthesis because of inactivation under some conditions
of' amino acid starvation (21). In a few cases, the aminoacyl-tRNA has been implicated in the repression of the cognate aminoacyl-tRNA synthetase. Thus, McGinnis and Williams have examined the role of tRNAhis in repression of' histidyl-tRNA synthetase formation in two strains of Salmonella typhimurium, a wildtype strain, and a tRNAhlS mutant (hisR) possessing only 52% of' the histidine acceptor activity of the wild-type strain (18). In both strains, histidyl-tRNA synthetase formation was derepressed during histidine-restricted growth. However, restoration of' histidine to the derepressed wild-type culture caused an immediate cessation of synthesis of' the enzyme, whereas the hisR mutant continued to synthetize the histidvl-tRNA synthetase at the derepressed rate despite the presence of' histidine. More recently, the same authors have investigated the involvement of histidyl-tRNA synthetase in regulating the expression ot' its own structural gene ( 17), and their results support the idea that histidyl-tRNA is the corepressor (or repressor) for histidyl-tRNA synthetase formation. In addition, in the case of methionyl-tRNA synthetase of' E. coli and S. typhimurium, it appears f'rom the examination of mutants of' this enzyme (4) that synthetase alteration(s) results in a derepression of synthesis of the enzyme due to a reduction in the acylation level of tRNAmet. However, the study of' the role of' aminoacyl-tRNA's in the regulation of aminoacyl-tRNA synthetases has proven difficult and laborious since it requires obtaining and characterizing mutants with altered synthetases or with altered tRNA's. In a previous report (9) we postulated that aminoalkyl adenvlates, considering their specif'ic action in vivo, should proxve usef'ul in investigations on the possible involvement of aminoacyl-tRNA's in regulation processes. This assertion is supported by the results presented in the present report. Indeed, it is shown that in E. coli K-12 a decrease in intracellular concentration of methionvl-tRNA induced by methioninyl adenylate leads to a specific derepression of the synthesis of methionyl-tRNA synthetase. Moreover, in the case of' strain HfrH, the relation between the level of methionyl-tRNA svnthetase and deacylation level of tRNAmet was determined: in cells grown in minimal medium, more than 25%, of tRNA-et is deacylated, methionyl-tRNA synthetase formation is derepressed and the level ot' derepression becomes proportional to the amount of tRNAmet deacylated. These results strongly support the hvpothesis that methionvl-tRNA is implicated
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METHIONYL-tRNA SYNTHETASE IN E. COLI K-12. II.
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