Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 11/28/14 For personal use only.
A new method for the isolation of methionyl transfer RNA synthetase mutants from Escherichia colil Department ofBiology, Ur~iversityyf Otta~vn,Otta~va,Coricrda K I N 6N5 Accepted February 3 , 1975
ARMSTRONG, J . B., and J . A. FAIRFIELD. 1975. A new method for the isolation of methionyl transfer R N A synthetase mutants from Eschericl~icrcoli. Can. J . Microbiol. 21: 754-758. Six methionine auxotrophs were isolated from an E. coli K-12 strain which required up to 100 times as much methionine for growth as a conventional auxotroph. In these mutants, the methionyl-tRNA synthetase had an increased K,,, for methionine. T h e K,,, value for the mutants ranged from 0.48 to 1.63 mM, compared to 0.078 mM for the wild type. T h e K,,, (methionine) for S-adenosyl methionine synthetase was not altered. ARMSTRONG, J. B.. e t J. A. FAIRFIELD.1975. A new method for the isolation of methionyl transfer RNA synthetase mutantsfrom Esclierichia coli. Can. J . Microbiol. 21: 754-758. A partir d'une souche de E. coli K-12 six auxotrophes a I'egard d e la methionine ont 6te isolis. Ces auxotrophes exigent jusqu'i 100 fois plus de methionine pour leur croissance qu'un auxotrophe ordinaire. Chez ces mutants, la methionyl-t ARN synthttase a unK,,, qui augmente pour la methionine. La valeur de K,,, pour les mutants varie de 0.48 h 1.63 mM, tandis que celle du type sauvage est de 0.078 mM. Le K,,, (methionine) pour la S-adenosyl methionine synthetase n'est pas change. [Traduit par le journal]
Introduction In genetic studies of many biochemical pathways, auxotrophic mutants can be isolated which require, for growth, the end product of a blocked reaction. If an end product, such as an aminoacyl-tRNA, cannot be taken up by the cells, or cannot be conveniently supplied for some other reason, mutant isolation is complicated. In some cases, information has come from the study of analog-resistant mutants. For the methionine pathway, ethionine-resistant mutants have included methionyl (met) tRNA synthetase mutants, with reduced affinity for the tRNA but with a normal K,,, for methionine (2), and S-adenosyl methionine (SAM) synthetase mutants (12, 13). Most aminoacyl-tRNA synthetase mutants, however, have been discovered among conventional auxotrophs. These have included not only met-tRNA synthetase mutants (14, 15), but also glycyl (10, 1l), tyrosyl (6), tryptophanyl (9), isoleucyl (l7), and histidyl (21) tRNA synthetase mutants. All show altered kinetic properties for the enzyme, with K,,, values for the amino acid substrates ranging up to several hundred times that of the wild type. The auxotrophic requirement of these mutants suggests that the increased requirement 'Received October 28, 1974.
of the aminoacyl-tRNA synthetase for the amino acid cannot be met by the endogenous pool. Consequently, it should be possible to select systematically for such mutants by looking for auxotrophs with a higher than normal amino acid requirement for growth. For the methionine pathway, such a procedure would seem equally likely to yield SAM synthetase mutants with an elevated K,,, for methionine. Transport mutants would not likely be isolated, as they would still be able to synthesize methionine, and should grow equally well at high or low methionine. Furthermore, Ayling and Bridgeland (5) found that a methionine auxotroph, carrying a transport defect leading to a K,,, for uptake two orders of magnitude greater than for the wild type, required no more methionine for growth than did a strain carrying the auxotro~hicmutation alone. In this paper we describe the isolation of mutants with a growth requirement for high methionine concentrations, all of which appear to be altered in met-tRNA synthetase.
Materials and Methods Bacteria Escherichia coli strain AW405 requires the amino acids threonine, leucine, and histidine for growth. Other properties of the strain have been described before (4). AW477 is a closely related strain which lacks the
ARMSTRONG AND FAIRFIELD: MET-tRNA MUTANTS FROM E. COLI
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 11/28/14 For personal use only.
histidine requirement, but is defective in NS,N1o-methyltetrahydrofolate reductase (metF) (3). Clietnicals L-Methionine-methyl-14C and 14C-adenosine triphosphate (U.L.) were obtained from Amersham Searle (Don Mills, Ontario) or New England Nuclear (Montreal, Quebec). Most other chemicals were obtained from the Sigma Chemical Company (St. Louis, Mo.). Transfer RNA was isolated from AW405 by the procedure of Zubay (24), or purchased from Grand Island Biological Co. (Grand Island, New York). Media The bacteria were grown on a minimal medium which contained, per litre of distilled water: K 2 H P 0 4 , 11.2 g; K H 2 P 0 4 , 4.8 g ; (NH4)2S04, 2.0 g; MgSO4.7H20, 0.25 g ; Fe2(S0,)3, 0.5 mg; glycerol, 10 g ; and thiamine, 1 mg. Required amino acids, unless otherwise specified, were added at a final concentration of 100 mgllitre. Plates containing minimal medium were prepared by adding 1.5x agar. Cultures were preserved by adding an equal volume of sterile glycerol to overnight cultures and storing at - 20C. Isolation of M~rrants The parental strain AW405 was grown on minimal medium containing M methionine ("high methionine") and mutagenized with N-methyl-N'-nitro-Nnitrosoguanidine according t o the procedure of Adelberg et al. (I). Overnight growth of the mutagenized cells on high methionine allowed for phenotypic expression. The bacteria were washed with 0.15 M NaCl and resuspended in medium with 1 0 - 4 M methionine ("low methionine") to an O D of about 0.05, measured in a n 18-mm-diameter tube with a Coleman Junior I1 spectrophotometer. The bacteria were grown for two generations, then penicillin G added to 3000 units/ml. Incubation was continued for 90 min, at which time the bacteria were washed again with 0.15 M NaCI, and suitable dilutions plated on high methionine plates. After growth, the colonies were replica-plated to low methionine plates. Those which failed to grow on low methionine were isolated and characterized as described below.
755
Purification of S A M Sytztherase The supernatant from the streptomycin sulfate precipitation step was brought to 4 5 x saturation with a saturated solution of (NH4),S04. The precipitate was removed by centrifugation, and the supernatant then brought t o 5 5 x saturation. The 5 5 2 precipitate was resuspended in Tris buffer and passed through a Sephadex G-25 column to remove residual (NH4),S0,. The enzyme was further purified on a DEAE4-Sephadex A-25 column. The enzyme eluted from the column at about 0.2 M NaCI. Etzzyme Assays SAM synthetase activity was measured by the procedure of Lombardini et al. (IS), which uses radioactive ATP.' The reaction is terminated by the addition of anion exchange resin to absorb the ATP, while the radioactive product SAM stays in solution. The only change made in the procedure was the use of uniformly labeled ATP instead of ATP-8-14C. Met-tRNA synthetase activity was assayed by measuring the rate of formation of 14C-met-tRNA by the procedure of Calendar and Berg (7). The procedure was altered by the substitution of 100 mM, pH 7.0, imidazole buffer for cacodylate and I mM dithiothreitol for 2-mercaptoethanol. Both commercial E. coli K-12 tRNA and tRNA prepared from the wild type (AW405) were used. The results were identical. Kinetic parameters were calculated according to the method of Wilkinson (22). Transport An exponentially growing culture was washed twice, by centrifugation, with lo-' M phosphate buffer, p H 7.0, then resuspended in the same buffer, containing 100 pg/ml chloramphenicol, t o lo9 cells/ml. After 10 min preincubation at 30C, 1-methionine-~nethyl-'~C was added. At intervals, 0.5-~nlsamples were removed, filtered rapidly through a 0.45-micron membrane filter (Millipore Filter Co., Montreal, Quebec), and washed with 10 ml 30C buffer. The filters were dried and counted in 10 ml of a scintillation solution consisting of 5 g 2,5-diphenyloxazole in 1 litre of toluene. Kinetic parameters were determined from the initial rate of uptake.
Preoaration o f Cell Extracts Results Bacteria were grown overnight in 500 ml of minimal ~ ~ M~~~~~~~ ~ ~ ~ medium in a 2800-ml Fernbach flask. The optical density The concentration Of an amino acid required was generally between 0.7 and 1.0. The bacteria were washed once, by centrifugation, with a buffer containing by an auxotroph undoubtedly reflects the 0.1 M T r i ~ pH , ~ 7.5, 1 mM EDTA,"nd 1 mM dithio- requirements of the cell, particularly for protein threitol. ~ h e ywere resuspended in the same buffer t o an synthesis. Providing, therefore, that the O D of about 20, and were disrupted by sonication. The system is not saturated, it should be possible sonicate was centrifuged at 10 000 rpm for 10 min in a to meet an Sorvall SS-34 rotor t o remove debris. A 1 0 x solution of fo adjust the exogenous streotomvcin sulfate was slowlv added t o the s u ~ e r n a t a n t Increased endogenous demand. The require. until a final concentration of 1 Z was reaclied, then ments of a methionine auxotro~h. ~ ~ 4 7 stirring was continued at 4C for 3 0 k i n . The preparation were met by 10-4 M methionineL(Fig. 1). A was recentrifuged at 18 000 rpm for 15 min, and the concentration of 10-2 M was chosen for the precipitate discarded. The supernatant was used without isolation of mutants with an increased requirefurther treatment for most of the enzyme assays.
i
A new method for the isolation of methionyl transfer RNA synthetase mutants from Escherichia colil Department ofBiology, Ur~iversityyf Otta~vn,Otta~va,Coricrda K I N 6N5 Accepted February 3 , 1975
ARMSTRONG, J . B., and J . A. FAIRFIELD. 1975. A new method for the isolation of methionyl transfer R N A synthetase mutants from Eschericl~icrcoli. Can. J . Microbiol. 21: 754-758. Six methionine auxotrophs were isolated from an E. coli K-12 strain which required up to 100 times as much methionine for growth as a conventional auxotroph. In these mutants, the methionyl-tRNA synthetase had an increased K,,, for methionine. T h e K,,, value for the mutants ranged from 0.48 to 1.63 mM, compared to 0.078 mM for the wild type. T h e K,,, (methionine) for S-adenosyl methionine synthetase was not altered. ARMSTRONG, J. B.. e t J. A. FAIRFIELD.1975. A new method for the isolation of methionyl transfer RNA synthetase mutantsfrom Esclierichia coli. Can. J . Microbiol. 21: 754-758. A partir d'une souche de E. coli K-12 six auxotrophes a I'egard d e la methionine ont 6te isolis. Ces auxotrophes exigent jusqu'i 100 fois plus de methionine pour leur croissance qu'un auxotrophe ordinaire. Chez ces mutants, la methionyl-t ARN synthttase a unK,,, qui augmente pour la methionine. La valeur de K,,, pour les mutants varie de 0.48 h 1.63 mM, tandis que celle du type sauvage est de 0.078 mM. Le K,,, (methionine) pour la S-adenosyl methionine synthetase n'est pas change. [Traduit par le journal]
Introduction In genetic studies of many biochemical pathways, auxotrophic mutants can be isolated which require, for growth, the end product of a blocked reaction. If an end product, such as an aminoacyl-tRNA, cannot be taken up by the cells, or cannot be conveniently supplied for some other reason, mutant isolation is complicated. In some cases, information has come from the study of analog-resistant mutants. For the methionine pathway, ethionine-resistant mutants have included methionyl (met) tRNA synthetase mutants, with reduced affinity for the tRNA but with a normal K,,, for methionine (2), and S-adenosyl methionine (SAM) synthetase mutants (12, 13). Most aminoacyl-tRNA synthetase mutants, however, have been discovered among conventional auxotrophs. These have included not only met-tRNA synthetase mutants (14, 15), but also glycyl (10, 1l), tyrosyl (6), tryptophanyl (9), isoleucyl (l7), and histidyl (21) tRNA synthetase mutants. All show altered kinetic properties for the enzyme, with K,,, values for the amino acid substrates ranging up to several hundred times that of the wild type. The auxotrophic requirement of these mutants suggests that the increased requirement 'Received October 28, 1974.
of the aminoacyl-tRNA synthetase for the amino acid cannot be met by the endogenous pool. Consequently, it should be possible to select systematically for such mutants by looking for auxotrophs with a higher than normal amino acid requirement for growth. For the methionine pathway, such a procedure would seem equally likely to yield SAM synthetase mutants with an elevated K,,, for methionine. Transport mutants would not likely be isolated, as they would still be able to synthesize methionine, and should grow equally well at high or low methionine. Furthermore, Ayling and Bridgeland (5) found that a methionine auxotroph, carrying a transport defect leading to a K,,, for uptake two orders of magnitude greater than for the wild type, required no more methionine for growth than did a strain carrying the auxotro~hicmutation alone. In this paper we describe the isolation of mutants with a growth requirement for high methionine concentrations, all of which appear to be altered in met-tRNA synthetase.
Materials and Methods Bacteria Escherichia coli strain AW405 requires the amino acids threonine, leucine, and histidine for growth. Other properties of the strain have been described before (4). AW477 is a closely related strain which lacks the
ARMSTRONG AND FAIRFIELD: MET-tRNA MUTANTS FROM E. COLI
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 11/28/14 For personal use only.
histidine requirement, but is defective in NS,N1o-methyltetrahydrofolate reductase (metF) (3). Clietnicals L-Methionine-methyl-14C and 14C-adenosine triphosphate (U.L.) were obtained from Amersham Searle (Don Mills, Ontario) or New England Nuclear (Montreal, Quebec). Most other chemicals were obtained from the Sigma Chemical Company (St. Louis, Mo.). Transfer RNA was isolated from AW405 by the procedure of Zubay (24), or purchased from Grand Island Biological Co. (Grand Island, New York). Media The bacteria were grown on a minimal medium which contained, per litre of distilled water: K 2 H P 0 4 , 11.2 g; K H 2 P 0 4 , 4.8 g ; (NH4)2S04, 2.0 g; MgSO4.7H20, 0.25 g ; Fe2(S0,)3, 0.5 mg; glycerol, 10 g ; and thiamine, 1 mg. Required amino acids, unless otherwise specified, were added at a final concentration of 100 mgllitre. Plates containing minimal medium were prepared by adding 1.5x agar. Cultures were preserved by adding an equal volume of sterile glycerol to overnight cultures and storing at - 20C. Isolation of M~rrants The parental strain AW405 was grown on minimal medium containing M methionine ("high methionine") and mutagenized with N-methyl-N'-nitro-Nnitrosoguanidine according t o the procedure of Adelberg et al. (I). Overnight growth of the mutagenized cells on high methionine allowed for phenotypic expression. The bacteria were washed with 0.15 M NaCl and resuspended in medium with 1 0 - 4 M methionine ("low methionine") to an O D of about 0.05, measured in a n 18-mm-diameter tube with a Coleman Junior I1 spectrophotometer. The bacteria were grown for two generations, then penicillin G added to 3000 units/ml. Incubation was continued for 90 min, at which time the bacteria were washed again with 0.15 M NaCI, and suitable dilutions plated on high methionine plates. After growth, the colonies were replica-plated to low methionine plates. Those which failed to grow on low methionine were isolated and characterized as described below.
755
Purification of S A M Sytztherase The supernatant from the streptomycin sulfate precipitation step was brought to 4 5 x saturation with a saturated solution of (NH4),S04. The precipitate was removed by centrifugation, and the supernatant then brought t o 5 5 x saturation. The 5 5 2 precipitate was resuspended in Tris buffer and passed through a Sephadex G-25 column to remove residual (NH4),S0,. The enzyme was further purified on a DEAE4-Sephadex A-25 column. The enzyme eluted from the column at about 0.2 M NaCI. Etzzyme Assays SAM synthetase activity was measured by the procedure of Lombardini et al. (IS), which uses radioactive ATP.' The reaction is terminated by the addition of anion exchange resin to absorb the ATP, while the radioactive product SAM stays in solution. The only change made in the procedure was the use of uniformly labeled ATP instead of ATP-8-14C. Met-tRNA synthetase activity was assayed by measuring the rate of formation of 14C-met-tRNA by the procedure of Calendar and Berg (7). The procedure was altered by the substitution of 100 mM, pH 7.0, imidazole buffer for cacodylate and I mM dithiothreitol for 2-mercaptoethanol. Both commercial E. coli K-12 tRNA and tRNA prepared from the wild type (AW405) were used. The results were identical. Kinetic parameters were calculated according to the method of Wilkinson (22). Transport An exponentially growing culture was washed twice, by centrifugation, with lo-' M phosphate buffer, p H 7.0, then resuspended in the same buffer, containing 100 pg/ml chloramphenicol, t o lo9 cells/ml. After 10 min preincubation at 30C, 1-methionine-~nethyl-'~C was added. At intervals, 0.5-~nlsamples were removed, filtered rapidly through a 0.45-micron membrane filter (Millipore Filter Co., Montreal, Quebec), and washed with 10 ml 30C buffer. The filters were dried and counted in 10 ml of a scintillation solution consisting of 5 g 2,5-diphenyloxazole in 1 litre of toluene. Kinetic parameters were determined from the initial rate of uptake.
Preoaration o f Cell Extracts Results Bacteria were grown overnight in 500 ml of minimal ~ ~ M~~~~~~~ ~ ~ ~ medium in a 2800-ml Fernbach flask. The optical density The concentration Of an amino acid required was generally between 0.7 and 1.0. The bacteria were washed once, by centrifugation, with a buffer containing by an auxotroph undoubtedly reflects the 0.1 M T r i ~ pH , ~ 7.5, 1 mM EDTA,"nd 1 mM dithio- requirements of the cell, particularly for protein threitol. ~ h e ywere resuspended in the same buffer t o an synthesis. Providing, therefore, that the O D of about 20, and were disrupted by sonication. The system is not saturated, it should be possible sonicate was centrifuged at 10 000 rpm for 10 min in a to meet an Sorvall SS-34 rotor t o remove debris. A 1 0 x solution of fo adjust the exogenous streotomvcin sulfate was slowlv added t o the s u ~ e r n a t a n t Increased endogenous demand. The require. until a final concentration of 1 Z was reaclied, then ments of a methionine auxotro~h. ~ ~ 4 7 stirring was continued at 4C for 3 0 k i n . The preparation were met by 10-4 M methionineL(Fig. 1). A was recentrifuged at 18 000 rpm for 15 min, and the concentration of 10-2 M was chosen for the precipitate discarded. The supernatant was used without isolation of mutants with an increased requirefurther treatment for most of the enzyme assays.
i
