JOURNAL OF BACrOLOGY, Oct. 1975, p. 140-148 Copyright i 1975 American Society for Microbiology

Vol. 124, No. 1 Printed in U.S.A.

Isolation of an Escherichia coli Mutant Deficient in Glutathione Synthesis' JAMES A. FUCHS AND HUBER R. WARNER* Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 Received for publication 11 July 1975

A mutant of Escherichia coli that contains essentially no detectable glutathihas been isolated. The mutant contains a very low level of the enzyme glutathione synthetase and accumulates y-glutamyl cysteine at a concentration approximately equal to the level of glutathione found in its parent. No significant differences in growth were observed between the mutant and its parent. However, the activity of at least one enzyme was found to be affected by the absence of glutathione; the specific activity of the B1 subunit of ribonucleoside diphosphate reductase was greatly reduced. The possibility that the decreased B1 activity is due to a mutation in the structural gene coding for B1 or its regulatory gene could be eliminated. This suggests that one role of glutathione in the cell is to maintain at least this one protein in an active state. We propose the designation gshB for the gene coding for glutathione synthetase.

one

The occurrence, chemical properties, metabolism, and biological functions of glutathione have been well summarized recently by Jocelyn (10). This almost universal constituent of living cells comprises up to 90% of the nonprotein thiols in mammalian cells and is also abundant in some bacteria, such as Escherichia coli. Although this compound has been implicated in y-glutamyl transpeptidase reactions, in the maintenance of protein sulfhydryl groups, in protection of cells from radiation, and as a coenzyme in some reactions, little is known about the specific roles of glutathione in the metabolism of bacteria. Some enzymatic reactions that require glutathione such as glyoxylase (EC 4.4.1.5) and formaldehyde dehydrogenase (EC 1.2.11) have even been referred to as metabolic curiosities. Diamide, a reagent that oxidizes sulfhydryl groups in erythrocytes (11), also temporarily inhibits E. coli cell division (28). Since the major nonprotein sulfhydryl compound in E. coli is glutathione, this suggests that reduced glutathione may be essential for cell division. The inhibitory effects of chloromethyl ketones on protein and ribonucleic acid synthesis and on cell division are reversed by reduced glutathione but not by other sulfhydryl compounds (21), suggesting that the effects of chloromethyl ketones may result from their specific reaction with intracellular glutathione. However, it is difficult to determine whether the oxidation of glutathione is the primary cause of ' Scientific Paper no. 9010 of the Minnesota Agricultural Experiment Station.

these inhibitory effects or merely a simultaneous occurrence. Many previous studies suffer from this ambiguity of interpretation. An approach to understanding the roles of glutathione in E. coli would be to isolate a mutant of E. coli unable to synthesize glutathione. In this paper we describe the isolation and partial characterization of such a mutant. MATERIALS AND METHODS Materials. [2- 4C ]glycine and D,L- [1- 14C ]a-amino-

n-butyric acid were obtained from Tracerlab and Calatomic, respectively. Both were purified by passage through a Bio-Rad AG-1-formate column to remove negatively charged impurities. [2,3-14C INethylmaleimide (NEM) was obtained from Amersham. [14C]cytidine 5'-diphosphate (CDP) and [1H JCDP were obtained from Schwarz/Mann. yGlutamyl-L-a-amino-n-butyric acid was a gift from William Rathbun. All other chemicals and enzymes were obtained from Sigma Chemical Co. Bacterial strains. The strains used in this study were all derived from E. coli S4,415 obtained from A. Munch-Peterson. This is an F- strain with the genotype metB, upp (defective in uridine monophosphate pyrophosphorylase [EC 2.4.2.9]), udk (defective in uridine kinase [EC 2.7.1.48]). This strain grows in the presence of either 10 tig of 5-fluorouracil or 5-fluorouridine per ml due to the absence of the above-mentioned enzymes. Growvth of bacteria. Cells grown in liquid medium were shaken in L-broth (13) or minimal medium (6), supplemented with methionine (30 ug/ml) and thymidine (100 Ag/ml) where appropriate. The cells were harvested when the absorbancy at 650 nm (A,,.) reached 1.0 (about 5 x 10' cells/ml) by centrifuging for 10 min at 10,000 x g. For isolation of single

140

VOL. 124, 1975

colonies or screening isolates on plates, L-broth agar or minimal medium agar, supplemented with thymidine (100 ug/ml), methionine (30 ,ug/ml), and cysteine (30 &g/ml) where appropriate, was used. Preparation of -y-glutamyl cysteine. A 15.5-mg amount of oxidized glutathione was dissolved in 1 ml of 0.2 M tris(hydroxynrethyl)aminomethane (Tris)chloride, pH 8.0, containing 0.1 M NaCl, and incubated at 37 C with 0.5 mg of carboxypeptidase A (EC 3.4.2.1) (41 U/mg). At various times 20-;il samples were removed, diluted with 1 ml of water, and heated for 2 min at 100 C. Portions (25 ul) of these heated samples were then assayed for glutathione content by using glutathione reductase (EC 1.6.4.2). The carboxypeptidase treatment was continued for 180 min, at which time the glutathione content had decresed to about 15% of its initial value. The entire reaction mixture was then incubated for 2 min at 100 C to inactivate the carboxypeptidase A. Reduced y-glutamyl cysteine was prepared by adding 10 ;mol of dithiothreitol to 5 ;&mol of oxidized 'y-glutamyl cysteine in 1 ml and letting the mixture stand at 4 C for 90 min. Preparation of extracts for Sephadex chromatography. Harvested cells were resuspended in (for G-75) or (for G-25) the original culture volume of 0.05 M Tris-chloride, pH 8, containing 10-' M ethylenediaminetetraacetic acid (EDTA), and these suspensions were treated five times for 1-min intervals, using a Bronwill Biosonik II sonic oscillator at 80% power. These suspensions were centrifuged at 5,000 x g for 10 min, and 0.2 volume of 5% streptomycin sulfate was added with stirring over a period of 15 min to precipitate nucleic acids. The precipitate was removed by centrifugation, and the supernatant fraction was placed in a boiling water bath for 3 min. The denatured protein was removed by centrifugation, and the supernatant fraction was chromatographed on Sephadex G-25 or G-75 as indicated

GLUTATHIONE-DEFICIENT E. COLI

141

potassium phosphate buffer, pH 7, containing 10-3 M EDTA, and the sulfhydryl content of this solution was then determined as indicated below. A slight excess of ["0C ]NEM was added to form the NEM derivatives of all sulfhydryl compounds present, and portions were chromatographed on Whatman no. 1 paper, with either n-butanol/formic acid/water (7:2:1) or 70% npropanol as the eluting solvent. These samples were co-chromatographed with standards prepared by adding a twofold excess of solid NEM to 0.01 M solutions of cysteine, -y-glutamyl cysteine, and glutathione. The standard derivatives were located with ninhydrin, and the radioactivity was located by radioautography and strip scanning on a Packard model 7201 radiochromatogram scanner. Preparation of thioredoxin, thioredoxin reductase, and glutathione reductase. Fractions A and B were prepared from E. coli KK1004 as described by Reichard (19). Thioredoxin was purified from fraction A as described by Laurent et al. (12). Thioredoxin reductase was purified from fraction B through the chromatography on Sephadex G-100 as described by Moore et al. (14), but omitting the ammonium sulfate step. This preparation was used for the experiments described in Fig. 1 and 2. We subsequently discovered that this preparation contained considerable glutathione reductase activity. Fraction B was also prepared from E. coli B/5 cells and chromatographed on diethylaminoethyl (DEAE)cellulose to separate the glutathione and thioredoxin reductases. Glutathione reductase was eluted slightly before thioredoxin reductase on the DEAE-cellulose column. The protein in those fractions containing glutathione reductase activity but free of thioredoxin reductase was concentrated by precipitation with ammonium sulfate. These preparations of thioredoxin, thioredoxin reductase, and glutathione reductase were stored frozen at -20 C in small portions until use. below. Assay of sulfhydryl, thioredoxin, and glutathiChromatography on Sephadex. Samples of 4 ml one content. An assay mixture containing 0.15 M were applied to a column (2.2 by 45 cm) of Sephadex Tris-chloride, pH 8.0, 0.05 M EDTA, 0.008% 5,5'G-75 in 0.02 M potassium phosphate buffer, pH 7, dithiobis-(2-nitrobenzoic acid) (DTNB), and 0.15 mg containing 10-8 M EDTA. Fractions of 5 ml were of reduced nicotinamide adenine dinucleotide phoscollected at the rate of about four fractions per hour. phate (NADPH) was prepared fresh each day. For the Portions (0.5 ml) of the fractions were added to 0.5 ml assay of sulfhydryl content in column fractions, 0.5 ml of thioredoxin reductase assay mix and assayed for of the fraction was added to 0.5 ml of the assay sulfhydryl content and for thioredoxin activity with mixture, and the A,1, was measured on a Beckman added thioredoxin reductase. Glutathione was also DB spectrophotometer after about 2 min. For the detected in this latter assay owing to contamination of assay of thioredoxin in the fraction, partially purified the thioredoxin reductase preparation with gluta- thioredoxin reductase was then added, and the rate of thone reductase. change of A,1, at room temperature was followed on Samples of 5 ml were applied to a column (2.5 by 43 a Sargent model SRL recorder. Due to contamination cm) of Sephadex G-25, fine, in 10-a M EDTA, pH 7.6. of the partially purified thioredoxin reductase prepaFractions (3.5 ml) were collected at the rate of about ration with glutathione reductase, the glutathione five fractions per hour. Fractions were assayed for content (reduced and oxidized) was also measured by sulfhydryl content and for thioredoxin and glutathi- the procedure used to determine thioredoxin in the one activity. column fractions. The rate of change was proportional Paper chromatography. Harvested cells were ex- to the amount of thioredoxin or glutathione present in tracted for 2 h at 0 C with 0.5% their original culture the fraction. volume of 5% trichloroacetic acid. The supernatant In vitro glutathione synthesis. Harvested cells fraction was then extracted five times with an equal were ground with twice their weight of alumina, and volume of ether to remove the trichloroacetic acid. this suspension was extracted with 4 volumes of 0.05 The extract was then diluted with 2 volumes of 0.3 M M Tris-chloride, pH 7.6, containing 10-a M EDTA.

142

FUCHS AND WARNER

This crude extract was centrifuged for 10 min at 12,000 x g, and the supernatant fraction was dialyzed overnight -against 100 volumes of the Tris-EDTA buffer. These dialyzed extracts were frozen until used in the following assays. For the assay of -y-glutamyl cysteine synthetase (EC 6.3.2.2) activity, assay mixtures containing 5 Mmol of adenosine 5'-triphosphate, 10 umol of Tris-chloride (pH 8.0), 5 Asmol of MgCl,, 2.5 umol of dithiothreitol, 5 umol of glutamic acid, 0.013 Amol of D,L- [1-_ 4C a-amino-n-butyric acid (2.4 x 107 counts/min per psmol), and enzyme in a total volume of 0.25 ml were incubated at 30 C. To stop the reaction, 2 ml containing 5 umol of unlabeled a-aminobutyric acid was added and the mixture was heated for 2 min in a boiling water bath. The sample was then applied to a column (3.3 by 50 mm) of Bio-Rad AG-1-HCO,-, and the column was eluted with 2 ml of water, 10 ml of 0.05 M NH4+HCO,-, and 3 ml of 1 M NH4+HCO,-. The radioactive product, ["C ]-y-glutamyl-a-aminobutyric acid, was eluted with 1 M NH4+HCO,-. A portion of this fraction was dried on a planchet and counted. For the assay of glutathione synthetase (EC 6.3.2.3) activity, assay mixtures similar to those above, except containing 0.5 umol of y-glutamyl-L-a-amino-nbutyric acid and 0.3 Mmol of ["C iglycine (8.4 x 10' counts/min per Mmol) in place of glutamic acid and D,L- ["C ]a-amino-n-butyric acid, were incubated with enzyme at 30 C in a total volume of 0.20 ml. The reaction was stopped as described above, but using 5 Amol of glycine instead of a-aminobutyric acid. The samples were applied to similar Bio-Rad AG-1HCO,- columns and eluted wiht 2 ml of water, 4 ml of 0.02 M triethylamine bicarbonate, pH 8 (TEA+HCO,-), 8 ml of 0.2 M TEA+HCO,-, and 5 ml of 1 M TEA+HCO,-. The radioactive product, yglutamyl-a-aminobutyryl glycine, elutes in the 1 M TEA+HCO,-. A portion of this fraction was dried on a planchet and counted. Ribonucleoside diphosphate reductase activity in ether-treated cells and crude extracts. Cells were grown in minimal medium supplemented with methionine, cysteine, and deoxyadenosine (50 Ag/ml) at 30 C with shaking. To an exponentially growing culture 5-fluorodeoxyuridine was added to give a final concentration of 20 ug/ml. At various times samples were removed and the cells were harvested and then treated with ether to make them permeable to nucleotides (26). These cells were assayed for ribonucleoside diphospate reductase (EC 1.17.4.1) activity as described by Warner (27). The reaction volume was reduced to 50 ;l, and the product and substrate were separated as described below. Thymine-starved E. coli B3 was grown in minimal medium (4), and crude extract was prepared and chromatographed on deoxyadenosine 5'-triphosphateSepharose as described by Fuchs et al. (9). The B2 activity present in the protein fraction which failed to adsorb to the column was chromatographed on DEAE-cellulose and hydroxyapatite (4), concentrated, and stored frozen until use. The Bi activity eluted by the adenosine 5'-triphosphate-containing buffer was also concentrated and stored frozen until

J. BACTERIOL. use. The Bi and B2 activity present in crude extracts was assayed in the presence of excess purified Bi or B2 activity as described by Fuchs et al. (9), with the following modifications. The reaction was stopped in the two assay procedures described above by heating the 50-Mgl reaction mixtures at 100 C for 2 min. Potato apyrase (EC 3.6.1.5) (6.25 gg) and deoxycytidine 5'-monophosphate (dCMP)(0.05 ,mol) were added to each assay tube, and the tubes were incubated for 30 min at 37 C to convert all nucleotides to nucleoside monophosphates. The tubes were again heated at 100 C for 2 min, and 20 ul of each reaction mixture (60 jAl total volume) was spotted on polyethyleneimine-cellulose plates. The plates were developed with 0.15 M LiCl saturated with boric acid and neutralized with ammonium hydroxide to separate dCMP from CMP. The area containing dCMP was then cut out and placed in a scintillation vial, and the nucleotide was eluted with 1 ml of 0.7 M MgCl, in 20 mM Tris-chloride, pH 7, for 30 min at room temperature. Ten milliliters of PCS solubilizer (Amersham/Searle) was added and the samples were counted in a Beckman LS-235 liquid scintillation spectrometer.

RESULTS The isolation scheme used was actually designed to isolate E. coli mutants deficient in either thioredoxin or thioredoxin reductase. These two proteins are required for the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates (4, 12, 14) but may also be involved in the reduction of sulfate to sulfite (17). Thus, the rationale was to select mutants that had simultaneously become defective in deoxyribose synthesis and cysteine synthesis. E. coli S0415 is deficient in both uridine monophosphate pyrophosphorylase (upp) and uridine kinase (udk) and thus is unable to convert 5-fluorouracil to a toxic ribonucleotide (2). Because of these mutations, the only pathway for utilization of 5-fluorouracil is the formation of 5-fluorodeoxyuridine from 5-fluorouracil and intracellular deoxyribose 1-phosphate. This nucleoside can then be phosphorylated to 5-fluorodeoxyuridylate, which is a potent inhibitor of thymidylate synthetase (5). E. coli Sk415, like all thymine prototrophs, has an extremely small intracellular pool of deoxyribose 1-phosphate and thus is unable to utilize this pathway. S4415 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (1), and temperaturesensitive thymine auxotrophs were isolated by selection for resistance to trimethoprim in the presence of thymidine at 42 C (15) and tested for 5-fluorouracil sensitivity at 30 C. One of the mutants, KK1004, that had simultaneously become thymine auxotrophic at 42 C but thymine prototrophic and 5-fluorouracil sensi-

GLUTATHIONE-DEFICIENT E. COLI

VOL. 124, 1975

tive at 30 C, was used as the parent in further studies. The growth of KK1004 is completely inhibited at 30 C in the presence of 5-fluorouracil (5 gg/ml), presumably because even at permissive temperature the defect in thymidylate synthetase greatly increases the intracellular pool of deoxyribose 1-phosphate, allowing 5-fluorouracil to be converted to 5-fluoro-

deoxyuridine. Derivatives of a mutagenized, phenotypically expressed culture of KK1004 that have regained resistance to 5-fluorouracil would be expected to include mutants defective in their ability to synthesize deoxyribose 1-phosphate due to a defect in reduction of ribonucleotides to deoxyribonucleotides, including those defective in thioredoxin and thioredoxin reductase. If thioredoxin and thioredoxin reductase are also required for the reduction of sulfate to sulfite for the ultimate synthesis of cysteine, thioredoxin and thioredoxin reductase mutants may be cysteine requiring. Therefore, the 5-fluorouracil-resistant mutants obtained were screened for cysteine auxotrophy at 30 and 42 C. E. coli KK1004 was grown in minimal medium at 30 C and mutagenized with N-methylN'-nitro-N-nitrosoguanidine as described by Adelberg et al. (1). The mutagenized cells were grown in L-broth at 30 C for three generations to permit phenotypic expression and then spread on minimal agar plates containing methionine and 5-fluorouracil (5 ug/ml). After incubation of the plates for 2 days at 30 C, 125 colonies were picked and streaked to obtain single colonies on L-agar, and these were tested for cysteine auxotrophy at 30 and 42 C on minimal agar. Mutants able to grow at 30 C in the absence of cysteine but able to grow at 42 C only in the presence of cysteine were selected for further study. To assay for thioredoxin activity in the mutants obtained, cell extracts were treated with streptomycin sulfate and the supernatant fractions were heated for 2 min in a 100 C water bath and then assayed with thioredoxin reductase purified through the first DEAE-cellulose column described by Moore et al. (14). The rate of reduction of the DTNB was dependent upon the amount of heated extract used and was presumed to be a measure of the amount of thioredoxin present. The relative rates of DTNB reduction obtained with the parent, KK1004, and the majority of the isolates assayed were 1.0 and 0.8 to 1.3, respectively. Two isolates, X88 and X101, gave background rates of DTNB reduction. We suspected that these isolates contained a heat-sensitive thioredoxin. Since strains X88 and X101 were isolated from the

143

same mutagenized, phenotypically expressed culture, they are probably progeny of the same original mutant. Unheated extracts of KK1004 and X88 and purified thioredoxin were chromatographed on Sephadex G-75 columns, and the fractions were assayed for A2,,, sulfhydryl content, and thioredoxin activity. The KK1004 and X88 extracts contained comparable amounts of thioredoxin activity eluting between 100 and 140 ml (Fig. 1). When the thioredoxin fractions from the KK1004 and X88 columns were heated for 2 min at 100 C there was a 20% decrease in thioredoxin activity in each sample. Thus, the mutant apparently does not have a heat-sensitive thioredoxin as initially suspected. The KK1004 extract contained a second component of actJivity that eluted between 140 and 180 ml and represented a compound (or compounds) of much lower molecular weight (Fig. 1). These fractions also contained considerable sulfhydryl groups. The X88 extract contained a similar sulfhydryl component, but no activity was associated with this component. Suspecting that the low-molecular-weight compound in the

a

E

0

.E

Ec 0

a) c'J 0 'o

c

IE I'5

0

40

120 80 160 ELUTION VOLUME,ml

200

FIG. 1. Sephadex G-75 chromatography of E. coli extracts. Extracts of E. coli KK1004 and X88 were prepared and chromatographed on Sephadex G-75. Portions (0.5 ml) of the fractions were assayed for A26,0 (0), sulfhydryl content (0), and thioredoxin and glutathione content (E).

144

J. BACEIOL.

FUCHS AND WARNER

KK1004 extract might be glutathione, we confirmed that the thioredoxin reductase preparation used to assay the column fractions was contaminated with glutathione reductase. The KK1004 and X88 extracts and glutathione were then chromatographed on Sephadex G-25 columns. The thioredoxin contents of the two extracts were comparable, whereas KK1004 but not X88 contained one or more lower-molecularweight compounds with enzymatic activity (Fig. 2). The two major components corresponded well with the elution volumes of oxidized and reduced glutathione. The major sulfhydryl component in the X88 extract has a slightly larger elution volume than that in the KK1004 extract, suggesting that the molecular weight of the sulfhydryl compound in the X88 extract is slightly less than that in the KK1004 extract.

20

30

FRACTION

60

m

FIG. 2. Sephadex G-25 chromatography of E. coli extracts. Extracts of E. coli KK1004 (O) and X88 (0)

prepared and chromatographed on Sephadex G-25, fine. Portions (0.5 ml) of the fractions were assayed for A,.0 (A), thioredoxin and glutathione content (B), and sulfhydryl content (C).

were

When ["IC ]NEM was added to trichloroacetic acid extracts of KK1004 and X88 to form ["C ]NEM derivatives of the low-molecularweight sulfhydryl compounds present and the products were separated by paper chromatography in n-butanol/formic acid/water, the major derivative (75%) present in the KK1004 extract corresponded to NEM-glutathione. A minor derivative (19%) with an Rf corresponding to that of NEM-y-glutamyl cysteine and an origin spot (6%) were also present. In contrast, the radioactive compounds in the X88 extract corresponded only to NEM-y-glutamyl cysteine (95%) and the origin (5%). Chromatography in 70% n-propanol confirmed that cysteinyl-glycine was not present in either extract. If the synthesis of glutathione in -E. coli is similar to that in liver (22) and other tissues and organisms, these results suggest that E. coli X88 may have a defective glutathione synthetase but that the y-glutamyl cysteine synthetase may be normal. This was confirmed by direct enzyme assays using crude extracts (Fig. 3). The specific activity of y-glutamyl cysteine synthetase was essentially identical in extracts of KK1004 and X88, whereas the glutathione synthetase activity in the X88 extract was only about 10% that found in the KK1004 extract. To verify that degradation of glutathione does not occur in the X88 extract, 0.2 Mmol of reduced glutathione was added to glutathione synthetase assay mixtures lacking glycine and y-glutamyl-L-a-amino-n-butyric acid and was incubated as usual. Portions were then assayed for glutathione content by using glutathione reductase. No loss of glutathione occurred in the presence of either KK1004 or X88 extracts, under conditions appropriate for the synthesis of 0.02 to 0.04 ,umol of glutathione. These results confirm that E. coli X88 is deficient in glutathione synthetase activity, presumably because of a mutation in the structural gene for glutathione synthetase. We propose the designation gshB for the gene mutated in X88 and X101. This mutation would explain the accumulation of y-glutamyl cysteine and the absence of glutathione in extracts of these mutants. It is not readily apparent why glutathionedeficient mutants would be isolated by screening procedures designed to select for a defect in deoxyribonucleotide biosynthesis. Thus, it was necessary to determine whether the cysteine auxotrophy and 5-fluorouracil resistance of X88 and X101 are related to the glutathione synthetase defect. First, KK1004, X88, and X101 were transduced to methionine prototrophy since methionine in the medium partially spares the cysteine requirement of X88 and X101, even

GLUTATHIONE-DEFICIENT E. COLI

VOL. 124, 1975 z

0.8

.

80 B

A

,0 X88 / KK1004

p0.6

KI1004 60

I0 cr

L

0OA

0,0

/

//

40

20.

° 0.24

2 0

-0_

I/. .

02 20

40

60

0

10 INCUBATION TIME minutes

20

-

--

X

30

FIG. 3. In vitro synthesis of glutathione. Extracts of E. coli KK1004 (0) and X88 (0) were assayed for y-glutamyl cysteine synthetase (A) and glutathione synthetase (B) activities by following the incorporation of D, L- ["4CJa-aminobutyric acid and [t4CJglycine into -y-glutamyl-a-aminobutyric acid and -y-glutamyl-

a-aminobutyryl-glycine, respectively.

145

deoxyadenosine (50 ug/ml) to the culture (3), and the reductase activity was measured at various times after addition. Whereas the activity in X88 increased to only 26 pmol/min per mg, the activity in KK1004 increased to 65 pmol/min per mg (Fig. 4). These data indicate that the ribonucleoside diphosphate reductase activity in X88 under derepressed conditions is less than one-half the activity in KK1004, and that X88 grown in minimal medium has a derepressed level of partially active enzyme. Similar results have been found for nrdB (8) and nrdA mutants (unpublished data); nrdA and nrdB are the structural genes for the Bi and B2 subunits, respectively, of ribonucleoside diphosphate reductase. To determine whether this difference in ribonucleoside diphosphate reductase activity is due to the different glutathione contents of KK1004 and X88, a gsh+ derivative obtained by P1 transduction was also assayed. This transductant had the derepressed level of activity found in KK1004, in contrast to the original mutant low level found in X88 (Table 1). A second derivative of X88, which had been transduced to nalA (nalidixic acid resistance) and gIpT (unable to use a-glyceryl phosphate), and thus contained the donor nrdA+ and nrdB+ genes which lie between the nalA and glpT

though methione is apparently not readily converted into cysteine in E. coli (20). The background growth in methionine-supplemented media makes selection of cysteine prototrophs more difficult. One derivative of X88, four derivatives of X101 that had spontaneously reverted to cysteine prototrophy at 42 C, and 23 P1-mediated transductants of X88 were selected and assayed for glutathione content. The glutathione content of these derivatives did not differ from that of their respective parents, indicating that the cysteine auxotrophy and the glutathione synthetase defect are independent KKI004 mutations in X88 and X101 and also that these z 60 mutations are not closely linked genetically. 0 To test whether the glutathione synthetase a. defect may be related to the 5-fluorouracil resistance of X88 and X101, as anticipated in the selection scheme, ether-treated cells of Z 40 KK1004 and X88 were assayed for ribonucleow side diphosphate reductase activity. Since the Bi protein of ribonucleoside diphosphate reduc11 I.. X88 tase is inactivated in vitro in the absence of D20 low-molecular-weight sulfhydryl compounds (4, aw 25), mutants deficient in glutathione such as X88 and X101 might have a reduced Bi activity 0 in vivo. Such a situation could reduce the o 0 c- a intracellular deoxyribose 1-phosphate pool, reLL sulting in reduced uptake of 5-fluorouracil. The ether-treated cells of KK1004 and X88 reduced 80 20 40 60 O CDP to dCDP at a rate of 16 and 12 pmol/min MINUTES AFTER Fd Urd ADDITION per mg of protein, respectively. Although this FIG. 4. Ribonucleoside diphosphate reductase acsuggests that X88 is not particularly deficient in tivity in ether-treated cells. E. coli KK1004 (0) and ribonucleoside diphosphate reductase activity, X88 (0) were grown in the presence of 5-fluorodeoxit is possible that the synthesis of ribonucleoside yuridine (FdUrd), and at various times after addition diphosphate reductase in X88 is less repressed of FdUrd portions were removed and the cells were compared with KK1004. To investigate this harvested. The cells were treated with ether and then possibility, both strains were derepressed by the assayed for ribonucleoside diphosphate reductase acaddition of 5-fluorodeoxyuridine (10 ,ug/ml) and tivity.

146

J. BACTERIOL.

FUCHS AND WARNER

TABLE 1. Ribonucleoside diphosphate reductase activity in ether-treated cells derepressed with 5-fluorodeoxyuridine E. coli strain

Relevant genotype

Activitya

KK1004 X88 X88 transductant X88 transductant

gshB+ gshB gshB+ gshB-naIA-nrdA+nrdB+-glpT

85 40 72 34

aPicomoles of dCDP formed per minute per milligram of protein.

genes (24), had the same low activity found in X88. This indicates that the reduced ribonucleoside diphosphate reductase activity in X88 is not due to a mutation in either the nrdA or nrdB gene. More conclusive proof that the specific activity of the Bi subunit is reduced owing to the glutathione deficiency depends upon a direct comparison of Bi and B2 subunit activities in the mutant and wild-type strains. In X101, both Bi and B2 protein activities were essentially unchanged by conditions that should give derepressed enzyme levels, i.e., growth in the presence of 5-fluorodeoxyuridine (Table 2). In contrast, in KK1004 significant derepression of both proteins was observed. Under repressed conditions the Bi protein activity in X101 was significantly less than that in KK1004, whereas the B2 protein activity was significantly higher in X101 than in KK1004. The ratio of Bi protein activity to B2 protein activity in X101 is thus less than one-tenth that in KK1004 both in the presence and absence of 5-fluorodeoxyuridine. These results indicate that the low ribonucleoside diphosphate reductase activity in X88 and X101 is not due to a defect in regulation of expression since both subunits of the enzyme are derepressed during normal growth. These results also support the hypothesis that glutathione deficiency causes inactivation of the Bi subunit of ribonucleoside diphosphate reductase, leading to derepression of the nrdA and nrdB genes. This could lead to limiting synthesis of deoxyribonucleotides in vivo, thus lowering the deoxyribose 1-phosphate pool and preventing the uptake of significant amounts of 5-fluorouracil. DISCUSSION Although not all experiments reported were done with both E. coli X88 and X101, these isolates behaved similarly in all experiments in which both were used. These include chromatography on Sephadex G-75 columns, growth

under various conditions, ribonucleoside diphosphate reductase assays, and various assays for glutathione activity. In addition to the glutathione defect, both X88 and X101 contained an unlinked temperature-sensitive defect in cysteine biosynthesis. Thus it is very likely that the two mutants are progeny from the same original mutant. The activity of y-glutamyl cysteine synthetase detected in KK1004 is less than 1% of the activity of glutathione synthetase detected. Since no attempt was made to optimize conditions for assaying either of these enzymes, the conditions for assaying -y-glutamyl cysteine may be very suboptimal. It is possible that -y-glutamyl cysteine synthetase is quite unstable or that a-aminobutyrate does not replace cysteine as efficiently in the y-glutamyl cysteine synthetase reaction as it does in the glutathione synthetase reaction in E. coli extracts. The large accumulation of y-glutamyl cysteine and the undetectable level of glutathione in X88 indicate that the in vitro difference between the specific activity of y-glutamyl cysteine synthetase and glutathione synthetase does not exist in vivo.

The glutathione deficiency does not inhibit growth significantly in either L-broth or minimal media. Thus, glutathione is not necessary for growth of E. coli under normal laboratory conditions. It seems likely that y-glutamyl cysteine might substitute for glutathione in some, but not all, of its roles. If glutathione is involved in amino acid transport in E. coli, as has been reported for mammalian tissues (16), -y-glutamyl cysteine appears to be able to replace glutathione as the source of y-glutamyl groups. The rat kidney transpeptidase is about one-half as active with y-glutamyl cysteine as it is with glutathione (23). Other reactions may specifically require glutathione. Glyoxylase converts methyl glyoxal to lactic acid by a two-step reaction requiring glutathione (18), and we have TABLE 2. Activity of ribonucleoside diphosphate reductase subunits in crude extracts y)prpnrplqv:..] a Liereprsseu

N-nntAArAnraeeAfl

E. coli

strain

-onaeURrepresse

Bi

B2

B1/B2

Bi

B2

B1/B2

KK1004 0.34b 0.08 4.3 1.44 0.68 2.1 0.08 0.26 0.3 0.06 0.31 0.2 X10o a Derepression was caused by the presence of 5-fluorodeoxyuridine (20 ulg/ml) and deoxyadenosine (50 tsg/ml) in the culture. Expressed as nanonioles of product formed per minute per milligram of protein.

VOL. 124, 1975

GLUTATHIONE-DEFICIENT E. COLI

found that X88 and X101 are more sensitive to methyl glyoxal than is KK1004 (unpublished data). This suggests that y-glutamyl cysteine cannot replace glutathione in detoxifying the methyl glyoxal via the glyoxylase reaction. Processes requiring merely the presence of a low-molecular-weight sulfhydryl compound should proceed as well in X88 and X101 as in KK1004, since the amount of y-glutamyl cysteine present in these cells is roughly equal to the amount of glutathione present in KK1004. The maintenance of Bi protein of the ribonucleoside diphosphate reductase enzyme in active form apparently does require glutathione since the specific activity of Bi protein is sharply reduced in X88 and X101. The ratio of oxidized and reduced lowmolecular-weight sulfhydryl compounds may be as important as the absolute amount of reduced sulfhydryl groups in maintaining the Bi subunit of ribonucleotide reductase in its active form. This possibility is presently being investigated. When glutathione forms a disulfide bond with any sulflhydryl group, the disulfide bond can be reduced by glutathione reductase and reduced nicotinamide adenine dinucleotide, as well as being able to undergo an exchange reaction. For a disulfide bond formed with -y-glutamyl cysteine only the latter reaction is possible. This difference could also explain the difference in Bi subunit-activity in X88 and KK1004. The observation that the concentration of low-molecular-weight sulfhydryl compounds in X88 and X101 is roughly equivalent to that in their parent is interesting since it implies that the glutathione reductase system is not necessary to maintain this concentration since oxidized glutamyl cysteine is not a substrate for glutathione reductase. It is possible that another system such as the "thioredoxin system" is important in maintaining the concentration of reduced thiols, at least under these conditions, or that an increased rate of y-glutamyl cysteine synthesis can keep the sulfhydryl content normal. Investigations are presently underway to answer some of these questions. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grants GM21015 and GM20884 from the National Institute of General Medical Sciences to H.R.W. and J.A.F., respectively. We also acknowledge support from the University of Minnesota Graduate School. H.R.W. is the recipient of Public Health Service career development award GM-45729 from the National Institute of General Medical Sciences. We thank Marianne Wright and Kathleen Lundberg for technical assistance, and William Rathbun for advice on the enzyme assays.

147

ADDENDUM IN PROOF P. Apontoweil and W. Berends have recently reported the isolation of E. coli mutants deficient in either 'y-glutamyl cysteine synthetase or glutathione synthetase (P. Apontoweil and W. Berends, Biochim. Biophys. Acta 399:10-22, 1975). All of their mutants grow normally in minimal media. Their -y-glutamyl cysteine synthetase mutant is more sensitive to methyl glyoxal and is not more sensitive to X irradiation, and its growth is more delayed by diamide than is its parent. Apontoweil and Berends did not discuss the properties of their glutathione synthetase mutant but we have made similar observations with our glutathione synthetase-deficient mutant. LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'nitro-N-nitrosoguanidine in Escherichia coli. Biochem. Biophys. Res. Commun. 18:788-795. 2. Beck, C. F., J. L. Ingraham, J. Neuhard, and E. Thomassen. 1972. Metabolism of pyrimidines and pyrimidine nucleosides by Salmonella typhimurium. J. Bacteriol. 110:219-228. 3. Biswas, C., J. Hardy, and W. S. Beck. 1965. Release of repressor control of ribonucleotide reductase by thymine starvation. J. Biol. Chem. 240:3631-3640. 4. Brown, N. C., Z. N. Canellakis, B. Lundin, P. Reichard, and L. Thelander. 1969. Ribonucleoside diphosphate reductase. Purification of the two subunits, proteins Bi and B2. Eur. J. Biochem. 9:561-573. 5. Cohen, S. S., J. G. Flaks, H. D. Barner, M. R. Loeb, and J. Lichtenstein. 1958. The mode of action of 5-fluorouracil and its derivatives. Proc. Natl. Acad. Sci. U.S.A. 44:1004-1012. 6. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B,,. J. Bacteriol. 60:17-28. 7. Fahey, R. C., S. Brody, and S. D. Mikolajczyk. 1975. Changes in the glutathione thiol-disulfide status of Neurospora crassa conidia during germination and aging. J. Bacteriol. 121:144-151. 8. Fuchs, J. A., and H. 0. Karlstrom. 1973. A mutant of Escherichia coli defective in ribonucleoside diphosphate reductase. 2. Characterization of the enzymatic defect. Eur. J. Biochem. 32:457-462. 9. Fuchs, J. A., H. 0. Karlstroim, H. R. Warner, and P. Reichard. 1972. Defective gene product in dna F mutant of Escherichia coli. Nature (London) New Biol. 238:69-71. 10. Jocelyn, P. C. 1972. Biochemistry of the SH group. Academic Press Inc.,. New York. 11. Kosower, N. S., E. M. Kosower, and B. Wertheim. 1969. Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem. Biophys. Res. Commun. 37:593-596. 12. Laurent, T. C., E. C. Moore, and P. Reichard. 1964. Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B. J. Biol. Chem.

239:3436-3444. 13. Luria, S. E., and J. W. Burrows. 1957. Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74:461-476. 14. Moore, E. C., P. Reichard, and L. Thelander. 1964. Enzymatic synthesis of deoxyribonucleotides. V. Puri-

148

15.

16. 17.

18. 19.

20.

21.

FUCHS AND WARNER

fication and properties of thioredoxin reductase from Escherichia coli B. J. Biol. Chem. 239:3445-3452. Okada, T., J. Homma, and H. Sonohara. 1962. Improved method for obtaining thymineless mutants of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 84:602-603. Orlowski, M., and A. Meister. 1970. The y-glutamyl cycle: a possible transport system for amino acids. Proc. Natl. Acad. Sci. U. S. A. 67:1248-1255. Porque, P. G., A. Baldesten, and P. Reichard. 1970. The involvement of the thioredoxin system in the reduction of methionine sulfoxide and sulfate..J. Biol. Chem. 245:2371-2374. Racker, E. 1951. The mechanism of action of glyoxylase. J. Biol. Chem. 190:685-696. Reichard, P. 1962. Enzymatic synthesis of deoxyribonucreotides. I. Formation of deoxycytidine diphosphate from cytidine diphosphate with enzymes from Escherichia coli. J. Biol. Chem. 237:3513-3519. Roberts, R. B., P. H. Abelson, D. B. Cowie, E. T. Bolton, and R. J. Britten. 1955. Studies of biosynthesis in Escherichia coli, p. 369-371. Carnegie Inst. Washington Publ. 607, Washington, D. C. Rossman, T., C. Norris, and W. Troll. 1974. Inhibition of macromolecular synthesis in Escherichia coli by pro-

J. BACTERIOL. tease inhibitors. J. Biol. Chem. 249:3412-3417. 22. Snoke, J. E., and K. Bloch. 1952. Formation and utilization of y-glutamylcysteine in glutathione synthesis. J.

Biol. Chem. 199:407-414. 23. Tate, S. S., and A. Meister. 1974. Interaction of -y-glutamyl transpeptidase with amino acids, dipeptides,and derivatives and analogs of glutathione. J. Biol. Chem. 249:7593-7602. 24. Taylor, A. L., and C. D. Trotter. 1972. Linkage map of Escherichia coli strain K-12. Bacteriol. Rev. 36:504-524. 25. Thelander, L. 1973. Physicochemical characterization of ribonucleoside diphosphate reductase from Escherichia coli. J. Biol. Chem. 248:4591-4601. 26. Vosberg, H., and H. Hoffmann-Berling. 1971. DNA synthesis in nucleotide-permeable Escherichia coli cells. I. Preparation and properties of ether-treated cells. J. Mol. Biol. 58:739-753. 27. Warner, H. R. 1973. Properties of ribonucleoside diphosphate reductase in nucleotide-permeable cells. J. Bacteriol. 115:18-22. 28. Wax, R., E. Rosenberg, N. S. Kosower, and E. M. Kosower. 1970. Effect of the thiol-oxidizing agent diamide on the growth of Escherichia coli. J. Bacteriol. 101:1092-1093.