Sep 8, 1976 - sine 5'-triphosphate [ATPI:thymidine 5'-phosphotransferase); tpp (deoA), thymidine phosphorylase (EC. 2.4.2.4, thymidine:orthophosphate ...
JOURNAL OF BACTERIOLOGY, Jan. 1977, p. 305-316 Copyright C 1977 American Society for Microbiology
Vol. 129, No. 2 Printed in U.S.A.
Thymidine-Requiring Mutants of Salmonella typhimurium That Are Defective in Deoxyuridine 5'-Phosphate Synthesis CHRISTOPH F. BECK,'* JAN NEUHARD, AND ELISABETH THOMASSEN Enzyme Division, University Institute of Biological Chemistry B, Copenhagen, Denmark Received for publication 8 September 1976
In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated. Thymine (thymidine)-requiring mutants, de- ence of thymine (thymidine). Phenotypically fective in thymidylate synthetase (EC 2.1.1b), such mutants would differ from thy mutants in are easily obtained in enteric bacteria by select- being capable of utilizing exogenous deoxyuriing for resistance to the folate analogue tri- dine as a source of thymidine nucleotides. methoprim in the presence of thymine (thymi- (Deoxyuridine is phosphorylated to DUMP by dine) (31). The specificity of the selection lies in thymidine kinase [EC 2.7.1.21] [Fig. 1; 26].) the nature of the thymidylate synthetase reacIt has been previously shown that in both tion, which is a stoichiometric transfer and re- Escherichia coli and Salmonella typhimurium, duction of a methylene group from N5,N1O- dUMP is synthesized via two independent pathmethylene tetrahydrofolate to deoxyuridine 5'- ways. The quantitatively more important pathphosphate (dUMP). The reductant in the reac- way involves the deamination of deoxycytidine tion (tetrahydrofolate) becomes oxidized to di- 5'-triphosphate (dCTP) to deoxyuridine 5'-trihydrofolate (25). To regenerate tetrahydrofo- phosphate (dUTP), followed by hydrolysis of late, which is required as a cofactor in numer- dUTP, probably by dUTPase (EC 3.1) (22, 24); ous other biochemical reactions; dihydrofolate Fig. 1), to dUMP. The dCTP deaminase (EC must be reduced by the nicotinamide adenine 3.5.4) from S. typhimurium has been purified dinucleotide phosphate-linked dihydrofolate re- extensively, and it was found to be a regulated ductase (EC 1.5.1.3), which is strongly in- enzyme; dCTP is a positive effector and deoxyhibited by trimethoprim. Thus, resistance to thymidine 5'-triphosphate (dTTP) is a negative trimethoprim in the presence of thymine occurs effector (2). The second pathway consists in if thymidylate synthetase is prevented from reduction of uridine 5'-diphosphate (UDP) by functioning (25). This condition is obviously ful- ribonucleotide reductase (EC 1.8) (25) to deoxfilled in mutants lacking significant thymidyl- yuridine 5'-diphosphate (dUDP), followed by ate synthetase activity (thy mutants). conversion of dUDP to dUMP (see Fig. 1). In the present paper we describe the isolation Mutants unable to synthesize dUMP should also be resistant to trimethoprim in the pres- and partial characterization of a S. typhimuI Present address: Department of Bacteriology, Univer- rium mutant defective in dUMP synthesis. As expected, two mutations are necessary to block sity of California, Davis, CA 95616. 305
306
BECK, NEUHARD, AND THOMASSEN
H*+ NADPH
NADP+
Thiored- S2
Thiored. - (SH)2
N
J. BACTROL.
u
I
j
| CDP, UDP
u l ur
* dTMP
dUMP
CdR - --*UdR
pj
TdR
deoxyribose -1 - P FIG. 1. Pyrimidine deoxyribonucleotide metabolism in S. typhimurium. The enzymes are identified by the corresponding gene designations, or by numbers, as follows: dcd, dCTP deaminase (EC 3.5.4, dCTP aminohydrolase); ndk, nucleoside diphosphate kinase (EC 2.7.4.6, nucleoside triphosphate:nucleoside diphosphate phosphotransferase); nrdA and nrdB, the two subunits of ribonucleoside diphosphate reductase (EC 1.8, reduced thioredoxin:ribonucleoside 5'-diphosphate oxidoreductase); thy, thymidylate synthetase (EC 2.1.1 .b, methylene tetrahydrofolate:dUMP C-methyltransferase); tdk, thymidine kinase (EC 2.7.121, adenosine 5'-triphosphate [ATPI:thymidine 5'-phosphotransferase); tpp (deoA), thymidine phosphorylase (EC 2.4.2.4, thymidine:orthophosphate deoxyribosyltransferase); cod, cytosine deaminase EC 3.5.4.1, cytosine aminohydrolase); cdd, cytidine (deoxycytidine) deaminase (EC 3.5.4.5, cytidine [deoxycytidine] aminohydrolase); 1, dUTPase (EC 3.1, dUTP diphosphohydrolase); 2, thioredoxin reductase (EC 1.6.4, reduced NADP:oxidized thioredoxin oxidoreductase); 3, thymidylate kinase (EC 2.7.4.9, ATP:thymidine 5'-phosphate [dTMPJ phosphotransferase); 4, uncharacterized phosphatase. The following abbreviations are used: C, cytosine; U, uracil; T, thymine; CdR, UdR, and TdR, the deoxyribonucleosides of C, U, and T; CDP and UDP, the 5'-diphosphates ofcytidine and uridine; dCDP, dUDP, and dTDP, the 5'-diphosphates ofdeoxycytidine, deoxyuridine, and thymidine, respectively; Thiored.-S2 and Thiored.-(SH)2, oxidized and reduced thioredoxin.
dUMP synthesis sufficiently to create a growth requirement for deoxyuridine. The two mutations are mapped on the S. typhimurium chromosome. One of the mutations causes a deficiency in dCTP deaminase activity. MATERIALS AND METHODS Media and growth conditions. AB medium (8) was used as minimal medium. In experiments where the nucleotide pools were determined,
tris(hydroxymethyl)aminomethane (Tris)-minimal
medium was used (10). Casamino Acids-enriched media are minimal media with 0.2% glucose and 0.2% Norite-treated, vitamin-free Casamino Acids (Difco). As complete medium, L-broth was used (6). Carbon sources were added to a final concentration of 0.2%, and growth factors were added in the following final concentrations (micrograms per milliliter): L-amino acids, 50; thiamine, 5; uracil, 10; hypoxanthine, 15; deoxyribonucleosides, 20. Solid media were prepared by adding 1.5% agar (Difco) to liquid media. For soft agar, 0.7% agar was added to the appropriate medium. Liquid cultures were grown with aeration on a rotary shaker at 37°C. Growth was monitored at 436
nm in an Eppendorf photometer model llOlM. One milliliter of bacterial culture with an absorbance of 1 at 436 nm contains 3 x 108 cells or 0.2 mg of dry
weight. Bacterial strains. All S. typhimurium strains used are derivatives of LT2. They are listed in Table 1, together with the E. coli strains used. Mutagenesis. N-methyl-N'-nitro-N-nitrosoguanidine was used for mutagenesis (13). Transductions. For transduction with Salmonella phage P22, the integration-deficient derivative L3 was used. Preparation of lysates and transductions were performed as described by Beck and Ingraham (3): For P1 transductions, Plvir was used. S. typhimurium strains used for P1 transductions must be galE in order to be sensitive to this phage (18, 27). Transductions as well as preparation of lysates were done according to procedures given by Enomoto and Stocker (11). galE mutants of S. typhimurium were obtained by selecting for resistance to phage FO (35; B. A. D. Stocker, personal communication). Mating procedure: time of entry experiments. Donor cultures were grown overnight without shaking at 37°C in L-broth containing 20 ,ug of thymidine
VOL. 129, 1977
Strain
S. typhimurJumLa JL-689 JL-891 KP-1289 KP-1361 KP-1363 KP-1365 KP-1367 KP-1372 KP-1385 KP-1389
S. TYPHIMURIUM DEOXYURIDINE-REQUIRING MUTANTS TABLz 1. Bacterial strains used Derived Genotype
307
Mode of construction
from:
HfrK5 metG319 trp ara-9 metG319 hisE35 galE strA HfrK4 serA13 argB69 lysA6 purI305 pyrF146 cdd-9 cod8 tpp-1 udp-11 dcd-1 b dum-1 argB69 lysA6 purI305 pyrF146 cdd-9 cod8 tpp-1 udp-11 KP-1363/F'15,011 arg+ thy+ lys+ argB69 lysA6 purI305 pyrF146 galE230 cdd-9 cod-8 tpp-1 udp-11 dum-1 KP-1361/F'15,01i arg+ thy+ lys+ argB69 lysA6 purI305 pyrF146 galE230 cdd-9 cod-8 tpp-1 udp-11 dcd-1 b dum-1 argB69 lysA6 purI305 pyrF146 galE230 cdd-9 cod-8 tpp-1 udp-11 dcd-1 Hfrh2 purE8 HfrK25 serA15 rfx argB69 lysA6 purI305 pyrF146 galE230 cdd-9 cod-8 udp-11 dcd-1 bdum-1 argB69 lysA6 purI305 pyrF146 cdd-9 cod8 tpp-1 udp-11 dum-1 argB69 lysA6 purI305 pyrF146 cdd-9 cod8 tpp-1 udp-11 dCd-1 argB69 lysA6 purI305 pyrF146 cdd-9 cod8 udp-11 dcd-1 b dum-1 argB69 lysA6 trp cdd-9 cod-8 tpp-1 udp-11 upp-24 dum-1 argB69 lysA6 trp cdd-9 cod-8 tpp-1 udp-11 upp-24 ded-1b argB69 lysA6 trp cdd-9 cod-8 tpp-1 udp-11 upp-24 dum-1 A[dcd-udk] argB69 lysA6 trp cdd-9 cod-8 tpp-1 udp-11 upp-24 dum-1 udk-13 lysA6 purI305 pyrF146 thy-1568 cdd-9 cod-8 udp-11 lysA6 purI305 pyrF146 thy-1568 cdd-9 cod-8 udp-11 dum-1 lysA6 purI305 pyrF146 thy-1568 cdd-9 cod-8 udp-11 dcd-1b
KP-1372
Reference 3 Reference 4 From J. L. Ingraham (JL-624) Arg-, Lys- segregant
KP-1363 KP-1385
Cross with E. coli SO-499 Cross with KP-1399
KP-1365 KP-1361
Described in text Resistance to phage FO
KP-1385
Cross with KP-1289
KP-1385
From J. Roth (SA-549) Same as SA-828 (30) Transduced tpp+ with P1
KP-1361
Transduced UdR+ with P22
KP-1361
Transduced UdR+ with P22
KP-1361
Transduced tpp+ with P22
KP-1412
P22 transductionc
KP-1413
P22 transductionc
KP-1420
Described in text
KP-1420
Resistance to FURd
KP-1363
P22 transductione
KP-1412
P22 transductione
KP-1413
P22 transductione
b
KP-1398 KP-1399 KP-1404 KP-1412
KP-1413
b
KP-1414
KP-1420 KP-1421 KP-1422
KP-1431
KP-1434
KP-1435 KP-1436 E. coli S0-88
SO-90 SO-139
SO-499
thi-1 rel-1 mal-24 spc-12 supE50 DE5(proB-lac)/F'128 pro+ lac+ metB1 rel-1 A[lac-purE]IF'13 lac+ purE+ thi-1 metE70 trpE38 purE42 proC32 leu-6 recAl mtl-l xyl-5 ara-14 lacZ36 azi-6 str109 tonA23 tsx-67 sup-45/F'254 lac+ purE+ thr leu thi thy lac pyr T1' strlF'15 arg+ thy+ Iys+
CGSC 4288' CGSC 5218f CGSC 4282'
From A. Eisenstark (KSU3852)
All S. typhimurium strains are derivatives of LT2. The designation for the gene encoding dCTP deaminase is dcd. The previous designation of this gene in E. coli was paxA (24), but it has been altered to dcd (23). c Derived in two transductional steps using P22 grown on appropriate donor strains: (i) purI+ upp- (3), followed by (ii) pyrF+ trp-. d FUR, 5-Fluorouridine (10 ,ug/ml). e Derived in two transductional steps using P22 grown on appropriate donor strains: tpp+, followed by a
b
(ii) argB+ thyf. -'Numbers from the E. coli Genetic St-ock Center (CGSC), Yale University, New Haven, Conn.
308
J. BACTERIOL.
BECK, NEUHARD, AND THOMASSEN
per ml. After a 20-fold dilution with the same medium, the cultures were incubated with slow shaking at 37°C. They were grown to a cell density of about 108 cells/ml. Recipients were grown overnight at 37°C in L-broth containing 20 ,ug of thymidine per ml with aeration. At 2-min time intervals, 108 donor cells and 4 x 108 recipient cells were mixed on filter membranes (Millipore Corp.; diameter, 25 mm). Excess liquid was removed, but care was taken to leave the membrane surfaces somewhat moist. These membranes were then placed on Lbroth agar plates (37°C), overlaid with 10 ml of Lbroth soft agar containing 20 ,ug of thymidine per ml. Plates with filters were incubated at 37°C. At indicated times the filters were transferred to tubes containing 2 ml of AB medium, and the mating was interrupted by vigorous shaking for 10 s; 0.1 ml of the suspensions or an appropriate dilution was added to 3 ml of AB medium soft agar and plated immediately on selective plates. Selection of recombinants. Recombinants that regained the ability to synthesize dUMP endogenously (UdR+) were selected for deoxyuridine and thymidine independence at 30°C. cdd+ recombinants were selected for ability to utilize deoxycytidine as the sole pyrimidine source at 37°C. tpp+ recombinants were selected for ability to utilize thymidine as the sole carbon source at 37°C. upp- recombinants were selected for resistance to 5-fluorouracil (10 ,ug/ml) at 37°C. udk- recombinants were selected for resistance to 5-fluorouridine (10 ,.g/ml) at 37°C. Transfer of episomes from E. coli to S. typhimurium. F' episome transfer was performed by spot tests on solid medium. The recipient S. typhimurium strain was mixed with the E. coli episome donor strain on selective minimal medium. The clones that arose were shown to be merodiploids by the appearance of segregants of the original phenotype. In transfers of F' lac episomes, merodiploids were selected on lactose as the sole carbon source. In transfers involving SO-88 as donor, T4 phages were added to the selective plates in order to kill the donor, since it has no markers for counterselection. Segregation of episomes. Small inocula of the merodiploids (104 cells) were grown overnight in 1 ml of L-broth containing 18 ,g of 5-aminoacridine; treated cultures were plated for single colonies on L-broth. Colonies were tested for the Lac phenotype, in the case of F'lac-containing merodiploids, or for the Arg,Lys phenotype in the case of the merodiploids containing the F'15 episome. Usually about 80 to 90% of the colonies had lost the episome. Nucleotide pools. Nucleotide pools were determined in cultures grown for at least two generations in liquid Tris medium in the presence of [32Plorthophosphate (specific activity in the medium, 10 iuCi/,l mol). Extraction and thin-layer chromatographic separation of the nucleotides has been described by Neuhard and Thomassen (21). Preparation of cell extracts. (i) Uridine kinase assays. A 40-ml amount of an exponentially growing culture in glucose AB medium was harvested, washed with Tris-hydrochloride, pH 7.8, 1 mM ethylenediaminetetraacetic acid (EDTA), and suspended in 1 ml of the same buffer (final cell density,
109 cells/ml). The suspensions were sonically treated for 1 min at 0°C in an MSE ultrasonic disintegrator and centrifuged; the supernatant was dialyzed for 2 h against 500 ml of the same buffer. (ii) dCTP deaminase and dUTPase assays. A 200ml portion of exponentially growing cultures in Casamino Acids-enriched AB medium was harvested, washed twice with 0.9% NaCl, suspended in 0.5 ml of 0.05 M potassium phosphate buffer (pH 6.8), 2 mM EDTA, 2 mM f8-mercaptoethanol (final cell density, 1012 cells/ml), and sonically treated for 1 min. Extracts were centrifuged, and the supernatant fluids were treated with 1/4 volume of 15% streptomycin sulfate at 0°C. After 30 min at 0°C the suspensions were centrifuged. In certain assays the supernatant fluid from the streptomycin step was dialyzed for 2 h against 500 volumes of the sonic oscillation buffer and used directly for assays. In other experiments (Table 5) the supernatant fluids were treated with solid ammonium sulfate to 55% saturation, left for 30 min at 0°C, and centrifuged. The precipitates were dissolved in 0.05 M potassium phosphate buffer (pH 6.8), 2 mM EDTA, 2 mM 8-mercaptoethanol and dialyzed against the same buffer for 2 h prior to assays. Ether treatment. Cells were made nucleotide permeable by ether treatment according to the procedure of Vosberg and Hoffmann-Berling (33). When stored deep-frozen, these preparations showed no loss of ribonucleotide reductase activity for months. Enzyme assays. For uridine kinase, dCTP deaminase, and dUTPase, one unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol of substrate per min at 37°C. Specific activities are expressed as units per milligram of protein, as determined by the method of Lowry et al. (16), using bovine serum albumin as standard. For ribonucleotide reductase, the specific activities are given in nanomoles of substrate converted at 30°C/ minute per 3 x 108 cells. Uridine kinase. Reaction mixtures of 40 ,Iu contained 0.05 M Tris-hydrochloride (pH 7.8), 2.5 mM MgCl2, 3 mM guanosine 5'-triphosphate, 1 mM [2'4C]uridine (specific activity, 0.5 ,uCi/,umol), and extract. Reactions were started by the addition of extracts and were performed at 37°C. At 5, 10, and 15 min, 10-,ul samples were applied to polyethyleneimine-impregnated cellulose thin-layer plates (28) and dried with hot air. The chromatograms were developed to 12 cm with water. After drying of the chromatograms, the areas containing phosphorylated uridine compounds (which did not migrate) were cut out, and the radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer.
dCTP deaminase. Activity was determined by spectrophotometric assay (2). In the experiments of Table 4, dialyzed streptomycin-treated extracts were used as enzyme sources. In the experiments reported in Table 5, ammonium sulfate-treated extracts were used. dUTPase. The radioactive assay described by Beck et al. (2) was used. However, the assay temperature was increased to 37°C. Streptomycin-treated extracts were used as enzyme sources. Ribonucleoside diphosphate reductase. The as-
S. TYPHIMURIUM DEOXYURIDINE-REQUIRING MUTANTS
VOL. 129, 1977
is a modification of the one described by Warner (34). Since nothing was known about the regulation of ribonucleoside diphosphate reductase of S. typhimurium, it was necessary, initially, to determine which effector gave the maximal enzymatic activity, with both UDP and cytidine 5'-diphosphate (CDP) as substrates. We found (results not shown) that 100 ,uM dTTP gave maximal activity with both substrates. With CDP as substrate, 100 ,uM dTTP stimulates the activity fivefold, and with UDP as substrate it stimulates the activity about 20-fold, over what is observed in the absence of any nucleoside triphosphate effector. We also observed that nicotinamide adenine dinucleotide phosphate (reduced) did not stimulate the activity in the presence of 40 mM dithiothreitol. The assay finally adopted contains, in a final volume of 50 ,ul, 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (pH 8.4), 8 mM MgCl2, 40 mM dithiothreitol, 100 ,uM dTTP, [3H]CDP (150 cpm/pmol) or ['4C]UDP (15 cpm/pmol), and ether-treated cells. The concentrations of substrates are given in the legend to Fig. 5. Reaction mixtures were incubated at 30°C. At 3, 5, 7, and 10 min, 10-,Il samples were withdrawn, applied directly to polyethyleneimine thin-layer plates, and dried with hot air. Prior to application, 10 nmol of each of the following nucleotides was added to each point of application as markers: uridine 5'-phosphate, dUMP, UDP, dUDP, uridine 5'-triphosphate, and dUTP in the case of UDP reduction assays, or the corresponding cytosine compounds when CDP reduction was assayed. Chromatograms were washed in absolute methanol for 5 min, dried, and developed ascending as follows: (i) absolute methanol (to the start line); (ii) 1 M ammonium acetate, 2.5% boric acid, pH 7.0 (with NH3) (to 2 cm above the start line); (iii) 2.5 M ammonium acetate, 3.6% boric acid, pH 7.0 (with NH3) (to 14 cm above the start line). In this system all six uridine nucleotides or all six cytidine nucleotides were separated from each other. The spots were located by ultraviolet light; the spots corresponding to the deoxyribonucleoside mono-, di-, and triphosphates were cut out, and the radioactivity was determined. Chemicals. The 5-fluoropyrimidine analogues were generously given to by A. von Sprecher (Hoffmann-La Roche). Trimethoprim, i.e., 2,4-diamino-5-(3-4,5-trimethoxybenzyl) pyrimidine, was the product of Burroughs Wellcome & Co.; 5-aminoacridine, nucleic acid bases, nucleosides, and nucleotides were obtained from Sigma Chemical Co. [2-'4C]uridine, [U-_4C]UDP (ammonium salt), [53H]dUTP (ammonium salt), and [5-3H]CDP (ammonium salt) were from the Radiochemical Center, Amersham, England. Carrier-free [32P]orthophosphate in 0.1 M HCl was from Atomenergikommissionens Forsogsstation, Riso, Denmark.
say
us
RESULTS
Selection of mutants defective to dUMP synthesis (UdR-). Mutants unable to synthesize dUMP from endogenous sources should be phenotypically thymine requiring (Thy-) and,
309
therefore, resistant to the folate analogue trimethoprim in the presence of thymine. However, they should differ from thy mutants (defective in thymidylate synthetase) in their ability to utilize deoxyuridine as a source of dTTP. In addition, such mutants should be sensitive to trimethoprim in the presence of deoxyuridine. The most frequent class of mutants obtained by the selection for trimethoprim resistance in the presence of thymine are mutants defective in thymidylate synthetase (thy). To avoid this class, we selected trimethoprim-resistant derivatives of strain KP-1365, which is diploid for the thy region of the chromosome because it harbors the E. coli episome F'15 (see Fig. 2). In addition, KP-1365 carries mutations in the structural genes for thymidine phosphorylase (tpp) (EC 2.4.2.4) and uridine phosphorylase (udp) (EC 2.4.2.3) and is thereby unable to catabolize deoxyuridine or thymidine (5). Additionally, it lacks cytidine (deoxycytidine) deaminase (cdd) (EC 3.5.4.5) and is thereby unable to synthesize deoxyuridine from deoxycytidine endogenously (5). A mutagenized culture of S. typhimurium KP-1365 was grown overnight in glucose minimal medium containing deoxyuridine (10 ,tg/ ml-) plus the necessary nutritional requirements uracil, hypoxanthine, and thiamine. Samples were plated on glucose-minimal plates containing thymidine (10 ,ug/ml) and trimethoprim (5 Ag/ml) in addition to the nutritional requirements of the strain. After 36 h at 370C resistant colonies were picked, purified, and further tested. One mutant, KP-1372, had acquired a growth requirement for thymidine that could be satisfied by deoxyuridine (UdR-) but was sensitive to trimethoprim in the presence of deoxyuridine. UdR- is not due to a mutation in thy. KP1372 was cured of its episome by treatment with 5-aminoacridine. All Arg-, Lys- segregants retained their deoxyuridine requirement, their resistance to trimethoprim in the presence of thymidine, and their sensitivity to trimethoprim in the presence of deoxyuridine. Thus, the UdR- phenotype of KP-1372 is independent of the F'15 episome. One of the segregants, KP1361, was kept for further studies. The sensitivity of KP-1361 to trimethoprim in the presence of deoxyuridine indicates that the strain contains a functional thymidylate synthetase. To verify that the UdR- phenotype is not a consequence of a modified thymidylate synthetase (a mutation in the thy gene), KP1361 was transduced Arg+, Lys+ with phage P22 propagated on an argB+ thy+ lysA + strain. All transductants retained the UdR- phenotype. Because most of them will have received the
310
J. BACTLRIOL.
BECK, NEUHARD, AND THOMASSEN
Hfr K2
d
wF15 nAi
H
K25
er
9ae Hfer K5 /
70
~~~~~~his
p -
tr
met G
\O
FIG. 2. Linkage rnap of S. typhimurium drawn according to Sanderson (29). The location of genes mentioned in the present work are shown. Closed arrows indicate the origin of the Hfr strains used; open arrows indicate the E. coli episornes used.
thy+ allele from the donor (see Fig. 2), the result indicates that the UdR- phenotype of KP-1361 is not a consequence of a mutation in the thy region of the chromosome. Growth behavior of KP-1372 and KP-1361. In liquid media supplemented with thymidine or deoxyuridine, the UdR- mutants KP-1361 and KP-1372 grew somewhat slower than their parent strains KP-1365 and KP-1362. This was so in glucose AB medium as well as in Casamino Acids-enriched medium (Table 2). In the absence of thymidine or deoxyuridine, KP-1372 underwent thymineless death (9). After removal of thymidine, cultures growing in Casamino Acids-enriched medium exhibited a lag of about 60 min before loss of viability was observed. Once initiated, death proceeded exponentially, with a half-life of about 60 min. This is a considerably slower death rate than observed with thy mutants starved for thymine (17), suggesting that the thymidine requirement of KP-1372 and KP-1361 is not absolute. The "leakiness" of the thymidine requirement of KP-1372 and KP-1361 was also apparent from their ability to grow slowly in the absence of thymidine or deoxyuridine on solid media with citrate or succinate as the sole carbon sources. With glycerol or glucose, however, no growth was observed in the absence of thymidine or deoxyuridine.
TABLE 2. Growth rates of strains of S. typhimuriuma Growth rate (dou-
blings/h)
Strain no.
KP-1363
KP-1365b KP-1361 KP-1372b
Relevant phenotype
UdR+ UdR+ UdRUdR-
G
G+ CAA
1.2 1.0 0.9 0.8
1.6 NDc 1.2 ND
a Cells were grown exponentially at 37°C in glucose AB medium containing: (G) arginine, lysine, uracil, hypoxanthine, thiamine, and thymidine; and
(G + CAA) Casamino Acids, uracil, hypoxanthine, thiamine, and thymidine. b KP-1365 and KP-1372 are isogenic with KP-1363 and KP-1361, respectively, except that the former contain the E. coli episome F'15. c ND, Not determined.
Acid-soluble nucleotide pools of KP-1372. The UdR- phenotype of KP-1372 suggests that it is defective in dUMP synthesis and, as a result, unable to synthesize thymidine nucleotides de novo. Thymine prototrophic strains of S. typhimurium contain very little dUMP in their acidsoluble pools (22). Howevei, blocking of thymidylate synthetase by either mutation (thy) or the addition of 5-fluorodeoxyuridine to the
S. TYPHIMURIUM DEOXYURIDINE-REQUIRING MUTANTS
VOL. 129, 1977
growth medium results in a large expansion of the dUMP pools of the cells (22). This is illustrated in Table 3, experiment 1, which shows that the addition of 5-fluorodeoxyuridine to KP-1365 resulted in depletion of the dTTP pool and a 50-fold increase in the dUMP pool of the cells. In contrast, such an increase in dUMP was not observed when KP-1372 was limited in dTTP by incubating the cells in thymidine-free medium in either the presence or absence of 5fluorodeoxyuridine (Table 3, experiment 2). Thus, the deoxyuridine requirement of KP-1372 seems to be the result of a defect in dUMP biosynthesis rather than a defect in conversion of dUMP to deoxythymidine 5'-monophosphate. Table 3 further shows that the accumulation of dCTP that accompanied the decrease in dTTP (see reference 21) was significantly more pronounced in KP-1372 than in the parent strain, KP-1365. Identification of the enzymatic defect in KP-1372 and KP-1361. It has been shown previously that 75% of the dUMP in S. typhimurium is derived from dCTP by the sequential action of dCTP deaminase and dUTPase (20, 22). Table 4 shows that the UdR- mutant KP-1372 (and derivative KP-1361) lacks detectable dCTP
311
TABLz 4. dCTP deaminase and dUTPase activities of mutants of S. typhimuriuma Sp act (nmol/min per mg of protein)
Strain KP-1363 KP-1365b KP-1361 KP-1372b
Relevant
phenotype UdR+ UdR+ UdRUdR-
dCTP deaminase
dUTPase
1.0 1.2
