Salmonella typhimurium: Role of N ... - Journal of Bacteriology

JOURNAL OF BACTERIOLOGY, May 1978, p. 528-536 0021-9193/78/0134-0528$02.00/O Copyright i 1978 American Society for Microbiology

Vol. 134, No. 2 Printed in U.S.A.

Arginine Auxotrophic Phenotype of Mutations in pyrA of Salmonella typhimurium: Role of N-Acetylornithine in the Maturation of Mutant Carbamylphosphate Synthetase AHMED T. H. ABDELAL,' ELIZABETH GRIEGO,' AND JOHN L. INGRAHAM2* Biology Department, Georgia State University, Atlanta, Georgia 30303,' and Department of Bacteriology, University of California, Davis, California 956162

Received for publication 6 December 1977

Mutations in pyrA that abolish catalytic activity of carbamylphosphate synthetase cause auxotrophy for both arginine and a pyrimidine. EightpyrA mutants auxotrophic only for arginine (AUX) were isolated by the mutagenized phage technique; three of these required arginine only at low temperature (20°C). Explanations of the AUX phenotype based on bradytrophy were eliminated by the discovery that blocking the utilization of carbamylphosphate for pyrimidine biosynthesis by insertion of an additional mutation in pyrB (encoding aspartic transcarbamylase) did not reduce the requirement for arginine. In contrast, mutational blocks in the arginine biosynthetic pathway before N-acetylornithine (argB, argC, argG, or argH) did suppress the mutation in pyrA. This suggests that exogenous arginine permits growth of the AUX mutants by inhibiting the first step in the arginine pathway, thereby preventing accumulation of an intermediate that antagonizes mutant pyrA function. A mutation in argA (N-acetylornithinase) failed to suppress AUX, indicating that N-acetylornithine was the inhibitory intermediate. This intermediate had no effect on the catalytic or regulatory properties of carbamylphosphate synthetase from mutant cells grown under permissive conditions (37°C). However, the regulatory properties of carbamylphosphate synthetase synthesized under restrictive conditions (20°C) were demonstrably defective (insensitive to activation by ornithine); the enzyme synthesized under permissive conditions was activated by ornithine. A strain carrying an additional mutation (argC), which prevents the accumulation of N-acetylornithine, produiced an ornithine-activatable enzyme at both growth temperatures. These results suggest that N-acetylornithine antagonizes the proper preconditioning or maturation of the mutant carbamylphosphate synthetase.

Carbamylphosphate is an intermediate in the biosynthesis of both arginine and pyrimidines. In Salmonella typhimurium (3) and Escherichia coli (13, 16, 20), it is synthesized from glutamine, bicarbonate, and ATP by a single enzyme, carbamylphosphate synthetase (CPSase), which is subject to cumulative repression by arginine and a pyrimidine compound and to dual feedback control: UMP inhibits activity and ornithine activates it. CPSase from S. typhimurium is similar in subunit structure to the enzyme from E. coli (20) in that: (i) it is composed of two unequal subunits (3); (ii) its heavy subunit alone catalyzes the synthesis of carbamylphosphate from ammonia, ATP, and bicarbonate, an activity that is stimulated by ornithine; and (iii) its light subunit confers on the heavy subunit the ability to utilize glutamine as the amide donor, and it confers sensitivity to feedback inhibition by UMP (3). 528

Mutations in the structural gene encoding CPSase (pyrA in S. typhimurium) that abolish catalytic activity cause auxotrophy for both arginine and pyrimidine. However, certain missense mutations in pyrA cause a variety of phenotypes whose biochemical bases are less obvious; these include arginine sensitivity (5), uracil sensitivity (2), and auxotrophy only for arginine (19, 23). Arginine auxotrophy of this type (AUX) confers the same phenotype as a strain that lacks ornithine transcarbamylase; it is such a frequent consequence of mutations within pyrA that it resulted in mismapping of the structural gene for ornithine transcarbamylase in S. typhimurium (19). The AUX phenotype has also been commonly observed in E. coli (16). The biochemical basis of the AUX phenotype remains unexplained. It has been suggested that it may result from leaky mutations (bradytrophy) in the structural gene for CPSase (7, 9, 23);

BASIS FOR ARGININE AUXOTROPHY IN pyrA

VOL. 134, 1978

i.e., the diminished supply of carbamylphosphate may be used preferentially to synthesize pyrimidines, thus generating a requirement for exogenous arginine (Fig. 1). This was not the case in the mutants we studied. Rather, exogenous arginine was required to inhibit the first step in the biosynthesis of arginine, thereby preventing the accumulation of an intermediate of the pathway, which antagonized the maturation of mutant CPSase. MATERIALS AND METHODS Cultures and culture techniques. All strains used in this investigation (Table 1) are derivatives of S. typhimurium LT2. The minimal salts medium used was that of Vogel and Bonner (21) to which, unless otherwise noted, amino acids were added to 50 jig/ml, and pyrimidine bases were added to 20 jg/ml. Transductional crosses were done as previously described

(6).

Preparation of cell extracts. Cells were harvested, washed once with water, suspended in 0.1 M potassium phosphate buffer (pH 7.6) containing 0.5 mM ethylenediaminetetraacetic acid and 1 mM phenylmethylsulfonylfluoride, and ruptured by passage through an Aminco French pressure cell. After centrifugation of the crude cell extract at 27,000 x g for 30 min, the supernatant was dialyzed for 3 h against 0.1 M potassium phosphate buffer (pH 7.6) containing 0.5 mM ethylenediaminetetraacetic acid. Protein concentration was determined by the method of Lowry et al. (11), with bovine serum albumin as a standard. Enzyme a8say8. CPSase activity was determiined in reaction mixtures (final volume, 0.5 ml) containing dialyzed extract, 100 mM triethanolamine buffer (pH 8.0), 12 mM ATP, 16 mM MgCI2, 10 mM NaH'4CQ3 (20,000 to 100,000 cpm/lpmol), 100 mM KCI, and 10 mM glutamine or 100 mM NH4CI. The ["4C]carbamylphosphate formed was determined as previously described (3). Ornithine transcarbamylase (OTCase) and aspartic transcarbamylase (ATCase) were assayed by determination of citrulline and carbamylaspartate as described by Prescott and Jones (18). The reaction mixture (final volume, 1 ml) for OTCase contained 100 mM triethanolamine buffer (pH 8.0), 2 mM ornithine, Glutamate

B C --

-

H

G

A

--4 a N-Acetyl- -

1 mM lithium carbamylphosphate, and enzyme. The reaction mixture (total volume, 1 ml) for ATCase contained 100 mM triethanolamine buffer (pH 8.0), 25 mM potassium aspartate, 4 mM lithium carbamylphosphate, and enzyme. For assay of the transcarbamylases, extracts were diluted serially in 0.05 M triethanolamine buffer (pH 8.0) containing 0.5 mM ethylenediaminetetraacetic acid and 0.05% bovine serum albumin. All determinations were made in the range in which reaction rates were constant and proportional to enzyme concentration. Chemicals. Amino acids, pyrimidines, and ATP were obtained from Sigma Chemical Co. Sodium [I4C]carbonate was purchased from New England Nuclear Corp. and used as a source of sodium bicarbonate in the reaction mixture.

RESULTS Isolation of mutants. Mutations in the structural gene encoding CPSase that cause arginine auxotrophy (AUX) have been described previously (4, 16, 19, 23). However, many of these mutants were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Therefore it is possible that the phenotype would be due to multiple mutations. To minimize this possibility, we isolated the mutants described here by using the mutagenized phage technique of Hong and Ames (8). P22 phage grown on the wild-type JL1 were treated with hydroxylamine under conditions that produced an average of one mutation per phage (8) and were then used to transduce to uracil independence strain JL1002, which carried a large deletion in pyrA, that covered regions encoding both subunits of the enzyme (3). Because our previous studies (2) have shown that the AUX phenotype is often more marked at lower temperatures of incubation, the transductants were scored for arginine auxotrophy at 20°C. Of 1,120 transductants picked, 8 required arginine for growth at 20°C. Five of these (JL2102, JL2103, JL2104, JL2105, and JL2107) also required arginine for growth at 37°C. The others (JL2101, JL2106,

Ornithine

Ornithine

529

I

Citrulline -*

).

Arginine

Glutamine +I CO2 + 2 ATP

pyr A

Carbamyl' phosphate

Aspartat

Carbamyl aspartate -. -+ pyr B

UMP

I~~~~~~~~~~~~~~~~~~~~~~~~~

uracil

FIG. 1. Pathways of biosynthesis of carbamylphosphate, arginine, and pyrimidines in S. typhimurium.

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ABDELAL, GRIEGO, AND INGRAHAM

J. BACTERUOL

TABLE 1. Strains of S. typhimnurium used in this investigation Genotype Phenotype Origin/source P. Hartman Prototrophic strain of LT-2 JL595 argB69 ARG-a K. E. Sanderson JL599 argG1O ARGK. E. Sanderson JL1002 pyrA81 ARG- pyR-b G. A. O'Donovan JL2101 pyrA1503 CS ARG-c Transduction of JL1002 with mutagenized phage JL2102 pyrA1504 ARGTransduction of JL1002 with mutagenized phage JL2106 pyrA1508 CS ARGTransduction of JL1002 with mutagenized phage JL2108 pyrA1510 CS ARGTransduction of JL1002 with mutagenized phage JL2109 pyrA1508 thy-1271 CS ARG- THYd JL2106, by resistance to trimethoprim with thymine JL2113 CS ARGJL2109 (see text) pyrAl5O8 JL2114 pyrA1508 argB69 ARGJL2109 (see text) JL2117 ARGargC113 K. E. Sanderson JL2119 argH95 ARGK. E. Sanderson JL2130 pyrA81 fol-181 ARG- PYR- TRIMe JL1002 by resistance to trimethoprim JL2171 pyrA81 fol-181 argH95 ARG- PYR- TRIM Transduction of JL2119 with phage grown on JL2130 JL2190 pyrA1510 argH95 ARGTransduction of JL2119 with phage grown on JL2108 JL2191 pyrA81 argCll3fol-181 ARG- PYR- TRIM Transduction of JL2117 with phage grown on JL2130 JL2192 ARG. J. Roth argA85 JL2201 pyrA1510 argC113 ARGTransduction of JL2191 with phage grown on JL2108 JL2204 pyrA81 argGlO fol-181 ARG- PYR- TRIM Transduction of JL599 with phage grown on JL2130 JL2205 pyrA1510 argGIO ARGTransduction of JL2204 with phage grown on JL2108 JL2211 pyrA81 argA85 fol-181 ARG- PYR- TRIM Transduction of JL2192 with phage grown on JL2130 JL2214 ARGpyrA1510 argA85 Transduction of JL2211 with phage grown on JL2108 JL2439 PYR- TRP-f TRIM Our culture collection pyrB tip JL2293 fol-181 Transduction of JL2130 with phage grown on JL1 JL2431 pyrA1503 fol-1 CS ARG- TRIM Transduction of JL2101 with phage grown on JL2293 JL2432 CS ARG- TRIM pyrA1504 fol-181 Transduction of JL2102 with phage grown on JL2293 a ARG-, Arginine auxotroph. b PYR-, Pyrimidine auxotroph. 'CS ARG-, Cold-sensitive prototroph at 37°C, arginine auxotroph at 200C. d THY-, Thymine auxotroph. ' TRIM, Resistant to 10 yg of trimethoprim per ml. f TRP-, Tryptophan auxotroph. Strain JL1

and JL2108) required arginine only at the lower temperature. For example, JL2108 grew at a near wild-type rate at 370C (doubling time of 65 min compared with 55 min for the parent strain); immediately after a shift to 200C in minimal medium, it grew at essentially the same rate as its parent (doubling times of 179 and 172 min for JL2108 and JL1, respectively), and then its specific growth rate declined gradually, reaching a

doubling time of 940 min after 16 h of growth at 200C. Effect of mutation on the pyrimidine pathway. The AUX phenotype suggests a specific deficiency of carbamylphosphate for use in the arginine pathway and a sufficiency for use in the pyrimidine pathway. To test this possibility, we assayed the levels of CPSase, OTCase, and ATCase produced by mutant strains. In all of


VOL. 134, 1978

531

has been reported to be suppressed by second mutations in pyrB (7). By means of the genetic blocking of the first step unique to the pyrimidine pathway, the resultant sparing of carbamylphosphate caused it to be used to synthesize arginine. Thus, the double mutant would require only pyrimidine for growth. In view of these data, we tested this hypothesis directly as follows. Two AUX strains, one of which (JL2101) requires arginine for growth only at 20°C, were made fol-181 by transductional crosses with JL2293 (fol-181) by using the latter as the donor and selecting for resistance to trimethoprim. The resulting strains, JL2431 and JL2432, respectively, were in turn used as donors in transductional crosses with JL2439 (pyrB), in which resistance to trimethoprim was selected. In the crosses JL2431 x JL2439 and JL2432 x JL2439, 31.9 and 20.4%, respectively, ofthe transductants CPSase. Effect of pyrB mutation on the AUX phe- required arginine for growth. In control crosses notype. The possibility that the AUX pheno- of JL2431 and JL2439 with JL1 (wild type), 20.7 type is the result of leaky mutations (7, 9, 12) and 27.8%, respectively, required arginine for growth. Thus, the pyrB mutation had no effect appears unlikely in view of the data presented in Table 2 and the similarity of the dissociation on the phenotype of these AUX mutants. Suppression of the AUX phenotype by constants for carbamylphosphate of OTCase (Abdelal et al., J. Bacteriol., submitted for pub- mutations in argB. Taken together, these data lication) and ATCase (17) (0.02 and 0.01 mM, show that exogenously supplied arginine was respectively). If carbamylphosphate were pro- required by AUX strains for any CPSase funcduced in suboptimal amounts, it would flow tion, rather than to compensate for inadequate approximately equally into the arginine and py- function. One consequence of supplying arginine rimidine pathways. Furthermore, the kinetics of exogenously was the inhibition of the activity of growth cessation upon shift of a cold-sensitive N-acetylglutamate synthetase, which catalyzed the first step of arginine biosynthesis, thereby arginine auxotroph from 37 to 20°C seems inconsistent with an explanation based on a lim- preventing accumulation of intermediates of the ited capacity to synthesize carbamylphosphate. pathway. Such inhibition was recently shown for If this were the case, the mutant growth would the purified enzyme from E. coli (12); a similar continue after a shift to 20°C only until the effect was demonstrated with a purified prepaintracellular pool was depleted and not at a ration of N-acetylglutamate synthase from S. diminishing rate for many generations, as was typhimurium (unpublished data). If feedback actually observed. However, an AUX phenotype inhibition of N-acetylglutamate synthase were

these AUX strains, ATCase was more derepressed than OTCase (Table 2). The maximal values (ratio of highest titer to lowest titer) are essentially the same (200-fold) for both ATCase and OTCase (6; A. T. H. Abdelal, E. H. Kennedy, and 0. Nainan, submitted for publication). In spite of the fact that exogenous arginine was present throughout most of the growth cycle, these results establish that the mutations caused starvation for the end products of both the arginine and pyrimidine pathways, thereby derepressing OTCase and ATCase. There was no direct correlation between CPSase levels as measured in vitro and the degree of derepression of OTCase and ATCase. For example, strain JL2107, which has relatively high levels of CPSase, produced higher levels of ATCase than did strain JL2106, which had only low levels of

TABLE 2. Titers ot CPSase, OTCase, and ATCase in various pyrA arginine auxotrophic mutants Sp acta (nmol/h per mg of protein) Strain

CPSase OTCase

ATCase

Ammonia JL1 (wild type) 160 34.6 (1) 35.5 5.55 (1) 27.7 19.4 207 (6) JL2101 132.5 (23.8) JL2102 24.5 53.9 35.7 (1) 21.8 (3.9) 12 13.1 JL2103 50.0 (1.4) 39.6 (7.1) 15.7 10 JL2104 232 (6.7) 135.5 (24.4) 16.1 JL2105 87.8 253 (7.3) 163 (29.3) 6.4 5.2 JL2106 235 (6.7) 49.5 (8.9) 77.3 6.1 JL2107 139 (3.9) 97.9 (17.6) 27.3 15.1 240 (6.9) JL2108 149.5 (26.9) a Cells were grown at 37°C on a limiting amount of arginine (10 ug/ml) and harvested, and extracts were prepared as described in the text. Numbers in parentheses indicate the number of fold derepression compared with that of the wild type. Glutamine

532


J. BACTERIOL.

the essential function of exogenous arginine that allowed growth of AUX mutants, a mutational block in the gene (argB) encoding N-acetylglutamate synthetase would permit growth in the absence of exogenous arginine. To test this hypothesis, we constructed a double mutant carrying both pyrA1508, which confers a cold-sensitive AUX phenotype, and argB69, which completely destroys N-acetylglutamate synthetase activity. This was accomplished as follows. By selection of spontaneous resistance to trimethoprim in the presence of exogenous thymine (10), a thy mutation was introduced into strain JL2106 (pyrAI508), generating JL2109 (pyrA1508 thy-1271), which in turn was used as a recipient in a transductional cross with JL595 ( argB69), selecting for thymine independence. Of the 200 THY' transductants, 22 were arginine auxotrophs at 370C as well as at 200C, indicating that they had inherited argB69 by transduction with thy-1271. Both ARG+ (JL2113 pyrA1508) and ARG- (JL2114 pyrA1508 argB69) transductants were selected for further study. That JL2114 did carry pyrA1508 was verified by transduction. This had to be established because strain JL2114 has the phenotype of one carryng argB69 alone. Phage grown on JL2114 were used to transduce JL1002 to uracil prototrophy at 370C. Of the 309 URA+ transductants, 283 were cold-sensitive arginine auxotrophs; i.e., pyrA1508 cotransduced 92% with pyrA81, establishing that JL2114 carried pyrA1508. Strains JL2113 (pyrA1508) and JL2114 (pyrA1508 argB69) were then compared in terms of their ability to synthesize carbamylphosphate and, hence, to grow at 200C (Fig. 2).

Neither mutant was able to grow on minimal medium (strain JL2113 grew slowly as a consequence of pyrA1508, and strain JL2114 failed completely to grow as a consequence of argB69); both strains grew at the wild-type rate with added arginine. However, these two strains differed markedly with respect to their response to ornithine; the single mutant JL2113 grew at the same low rate as in unsupplemented medium, but the double mutant JL2114 grew at the wildtype rate. Carbamylphosphate is required for ornithine to be converted to arginine. Thus, growth in the presence of ornithine established that a normal supply of carbamylphosphate was available in the double mutant; i.e., the argB69 mutation completely suppressed the pyrA1508 mutation. Suppression of AUX by other arg mutations. The suppression of pyrA1508 by argB69 can be explained in one of two ways: (i) The argB product itself is required for proper functioning of the mutant form of CPSase in a situation analogous to the arginine-sensitive mutations in pyrA that require OTCase to assemble mutant CPSase (5), or (ii) an intermediate of the arginine pathway inhibits function of the mutant enzyme. To distinguish between these two possibilities, double mutants carrying pyrA1510 as well as mutations in other genes of the arginine pathway were constructed. JL2108 (pyrA1510), which has a phenotype essentially identical to that of JL2113, was selected for these constructions because it possesses a significant titer of CPSase, a property that was desirable for subsequent enzymatic studies. All double mutants were constructed by the route described for the strain carrying pyrA1510 and argC113.

0.5

Ec 0

N

NO ADDITIONS

+ORNITHINE

+ARGININE

0.4

0.3~

U

z

0.2k

m r 0 (A

0t 0.1 100

200

300

400

100

200

300

400

100

200

300

400

TIME (MIN.)

FIG. 2. Growth of (A) JL2113 (pyrA1510) and (@) JL2114 (pyrA1508 argB69) at 20°C in mineral salts medium (A) and in the same medium supplemented with 100 pg of ornithine per ml (B) and with 100 pg of arginine per ml (C). Strains were grown in the same medium at 20°C for 16 h before absorbance was measured. During the first 16-h growth period at 20°C, the culture grew at a constantly decreasing rate (see text).

VOL. 134, 1978


Strain JL1002, which carries the large deletion pyrA81 and therefore requires both arginine and uracil to grow, was made fol by selecting for spontaneous resistance to 10 ,ug of trimethoprim per ml (10). To take advantage of the 18% contransduction frequency between fol and pyrA, we introducedpyrA81 and fol-181 in the arginine auxotroph (e.g., argC113) by selecting for resistance to 4 ,g of trimethoprim per ml and by scoring the transductants for arginine and uracil requirements. Then, by transduction, the pyrA81 mutation was replaced by pyrA1510 in a cross in which JL2108 was the donor; clones that grew in the presence of arginine and trimethoprim were kept. By the route outlined above, double mutants between pyrA1510 and argCl13, argH95, argG10, or argA85 were constructed. In each case, genotypes were verified by two kinds of transductional crosses, with the double mutant as the donor. In the first kind, in which uracil independence was selected, JL1002 (pyrA81) was used as a recipient; in each of these crosses, over 90% of the transductants required arginine for growth at 200C, thus establishing that the double mutants did indeed carry pyrA1510. In the other kind of cross, strains carrying argC13, argH95, argG10, and argA85 mutations were used as recipients; proof that the double mutant did carry the proper arginine mutations was inferred from the fact that crosses with the homologous recipient resulted in no arginineindependent transductants, whereas normal numbers of transductants were obtained in crosses with heterologous recipients. The phenotypes of the various double mutants are summarized in Table 3. Mutations in argB (as discussed previously), argC, argH, and argG suppressed the phenotype of pyrA1510. The mutation in argA did not. Thus it appears that no single protein of the arginine pathway interacted with mutant CPSase to cause the AUX phenotype. Rather, we propose that the phenotype was the result of the effect of an intermediate of the pathway on mutant CPSase. On the basis of the finding that argB, argC, argH, and argG mutations suppressed and argA85 did not, we can conclude that the intermediate was N-acetylornithine (Fig. 1). Effect of N-acetylornithine on mutant growth. N-acetylornithine did not inhibit the growth of JL2108 (pyrA1510) growing at 370C, as might be expected; neither did it inhibit the growth of any of the double mutants growing at 200C in the presence of ornithine. However, exogenous N-acetylornithine did not swell the intracellular pool, as indicated by finding that, at a concentration of 200 ,ug/ml, it supported the

533

TABLE 3. Response of various double mutants to ornithine and arginine at 20°C

Strain

Genotype

Growtha on minimal medium with: No addi- Orni- Argitions

thine

nine

+ JL2113 pyrAI508P + + JL2114 pyrA1508 argB69 + + JL2201 pyrA1510 argC13 + + JL2190 pyrA1510 argH95 + + JL2205 pyrA1510 argGlO + JL2214 pyrA1510 argA85 a On plates scored after 4 days at 20°C. +, Growth; -, no growth. b The phenotypes of pyrA1508 and pyrA1510 are the same (see text). On plates, no growth was visible on minimal medium or minimal medium supplemented with ornithine after 4 days in spite of the fact that slow growth occurred in liquid forms of these media (Fig. 2). The reasons for this apparent difference are not clear.

growth of auxotrophic mutants at a rate less than half that of the parent strain. This restrictive utilization of N-acetylornithine is similar to that reported for E. coli (22). Effect of N-acetylornithine on mutant CPSase. The effect of N-acetylornithine on mutant CPSase was examined at a nonsaturating concentration of ATP, because previous studies (3) have shown that all effectors of the S. typhimurium enzyme exert their action by altering the affinity of the enzyme for ATP. N-acetylornithine at a concentration of 5 mM stimulated CPSase activity severalfold in extracts of JL1 (wild type) and JL2108 (pyrA1510) (data not shown). Because this compound has no effect on purified CPSase from strain JL1, the stimulation observed in crude extracts was presumed to be the result of derepressed levels of N-acetylornithinase, which converted N-acetylornithine to ornithine, a known activator of the enzyme. Accordingly, we examined the effect of N-acetylornithine in extracts of JL2192 (argA85) and JL2214 (pyrA1510 argA85), which lack Nacetylornithinase activity. This compound did not modify CPSase activity in either the presence or the absence of allosteric effectors (Table 4). Effect of growth temperature on the regulatory properties of mutant CPSase. The absence of any effect of N-acetylornithine on the activity of mutant CPSase, together with the slow, progressive decline of the growth rate of cold-sensitive arginine auotrophs upon transfer to restrictive conditions, suggested that the effect of N-acetylornithine might be on the mat-

534

J. BACTERIOL.


TABLE 4. Effect of N-acetylornithine on CPSase activity in extracts of JL2192 (argA85) and JL2214 (pyrA1510 argA85) in the absence or presence of effectors Carbamylphosphate formed (nmol/10 min)' JL2214

Effector

JL2192

(pyrAI51OargA85)

10 mM Glutamine

0.2 mM Glutamine

0.2 mM Glutamine

14.7 1.3 7.4 None 14.9 7.7 1.2 N-acetylornithine (5 mM) 21.1 Ornithine (0.01 mM) 23.5 Ornithine (0.01 mM) + N-acetylornithine (5 mM) 51.4 2.0 28.1 Ornithine (0.1 mM) 2.1 53.7 28.0 Ornithine (0.01 mM) + N-acetylornithine (5 mM) 8.4 1.1 3.1 UMP (0.1 mM) 9.9 1.1 3.4 UMP (0.1 mM) + N-acetylornithine (5 mM) 56.5 UMP (0.1 mM) + 0.4 mM ornithine (0.4 mM) 56.1 UMP (0.1 mM), omithine (0.1 mM) + N-acetylomithine (5 mM) a Enzyme was assayed as described in the text except that the concentrations of ATP and MgC12 were 3 and 7 mM, respectively, and the concentration of glutamine was varied as indicated.

uration or the assembly of the mutant enzyme. If this hypothesis were correct, the properties of CPSase from cells grown under restrictive conditions might be different from those grown under permissive conditions. To test this hypothesis, we grew cells of JL2108 (pyrA1510) and JL2201 (pyrA1510 argC113) at 37 and 200C and assayed their extracts at both temperatures in the presence and absence of effectors. The activity of mutant CPSase in extracts of JL2108 grown under permissive conditions was highly stimulated by ornithine at both 37 and 20°C (Table 5). In contrast, the enzyme from JL2108 grown under restrictive conditions (200C, no added arginine) was not stimulated by ornithine at either 37 or 200C. As a control, JL2108 was grown at 200C in the presence of arginine; the enzyme produced under these particular permissive conditions is also normal with respect to activation by ornithine (unpublished data). Unlike the wild-type enzyme (3), CPSase from mutant cells grown under permissive or restrictive conditions was only slightly inhibited by UMP. Preliminary data (not shown) indicate that the enzymes produced under restrictive and permissive conditions also have different kinetic properties with respect to concentrations of substrate. If the difference in regulatory properties of the enzyme obtained from cells grown under both conditions were a consequence of the effect of N-acetylornithine on the assembly or maturation of CPSase, strains harboring secondary mutations that suppress pyrA1510 would produce an enzyme with similar properties, regardless of growth temperature. This hypothesis was tested with strain JL2201 (pyrA1510 argC113). The results (Table 5) show that this was the

TABLE 5. Regulatory properties of CPSase in extracts of JL2108 (pyrA1510) and JL2201 (pyrA1510 argC113) grown at 37 or 20°C Strain Growtha (OC)

Addition to reaction

mixture" JL2108

37

None

Ornithine 20

UMP None

Ornithine UMP None

Sp act (nmol/h per mg of protein) 370C

6.5 35.5 5.1 162 178 142

(100) (546) (78) (100) (110) (87)

200C

3.6 (100) 19.4 (539) 3.0 (83) 42.6 (100) 44.8 (105) 44.2 (104)

(100) 27.9 (100) (190) 66.5 (238) UMP (71) 21.2 (76) 20 None (100) 15.4 (100) Ornithine (245) 37.8 (245) UMP 12.8 (83) (83) a Cultures were grown on glucose minimal medium supplemented with ornithine at 10 ug/ml. Cultures were initially grown at 370C to an optical density of 0.2 at 420 nm. The cultures were split. One part was grown at 37°C and the other was grown at 200C. Both cultures were harvested at an optical density of 0.8 at 420 nm, and extracts were prepared as described in the text. h Dialyzed extracts were assayed as described in the text, except that the concentrations of ATP and MgCl2 were 2 and 6 mM, respectively. Ornithine and UMP, when present, were at 0.02 and 0.1 mM, respectively.

JL2201

37

Ornithine

62 117.5 44.3 17.3 42.4 14.4

case; CPSase activity from cells grown at 37 and 20°C was highly stimulated by ornithine.

DISCUSSION Although complete loss of CPSase activity causes auxotrophy for both arginine and pyrimidine, missense mutations in pyrA (the gene encoding this enzyme) can lead to a variety of

VOiL. 134, 1978


unexpected phenotypes. The biochemical basis of one of these, the arginine-sensitive phenotype, has been described recently (5). In this paper, we have presented evidence relating to the biochemical nature of the arginine-auxotrophic (AUX) phenotype. It has been suggested that the AUX phenotype is a consequence of bradytrophy; i.e., a limited supply of carbamylphosphate is preferentially used to synthesize pyrimidines (7, 9, 12). In the present work, we show that bradytrophy cannot account for the phenotype ofthe mutants we studied. Introduction into such a strain of a mutation in pyrB (the gene encoding aspartate transcarbamylase), which utilizes carbamylphosphate in the pathway leading to pyrimidine biosynthesis, does not eliminate the requirement for arginine by the strain. Were bradytrophy the explanation, insertion of a pyrB mutation would produce a strain requiring only pyrimidine, because all carbamylphosphate would be available for the synthesis of arginine (the demands for arginine and for pyrimidine by the cell are approximately equal). Rather, the arginine auxotrophy of these mutations in pyrA was related to inhibition by arginine of the first step of the arginine pathway because insertion of an argB mutation restored normal function of the mutant CPSase. This suppresson could indicate that either the argB product itself or an intermediate of the arginine pathway before ornithine inhibited the function of the mutant enzyme. Double mutants carrying both pyrA1510 and mutations in the arginine pathway, including argC, argG, and argH, were also suppressed for pyrA function; these results show that an intermediate of the arginine pathway prevented mutant pyrA function. The failure of a mutation in argA to suppress indicates that the inhibitory intermediate was N-acetylornithine. We were unable to demonstrate any effect of N-acetylornithine on the catalytic or regulatory properties of the mutant enzyme in cell extracts. This fact, along with the slow, progressive decline in the growth rate upon transfer to restrictive conditions, suggests that N-acetylornithine affects the maturation or assembly of the enzyme. This hypothesis is supported by the finding that mutant cells (pyrA1510) produced an enzyme with altered regulatory properties under restrictive conditions but not when the strain carried a second mutation (argC113) that blocked the formation of N-acetylornithine. We do not suggest that altered regulatory properties per se are the total explanation for the lack of functioning of the enzyme; the enzyme synthesized under restrictive conditions was also al-

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tered with respect to other important parameters, including a high Km for ATP (unpublished data). Paulus and Alpers (15) have cited examples of small molecules that enhance the rate of maturation of oligomeric enzymes from a relatively unstable form to a stable one, and they called this structural transition "preconditioning." A possible explanation for the effect of Nacetylornithine on the mutant carbamylphosphate synthetase is antagonism of the preconditioning step. Such an antagonism would become more critical at lower temperatures (restrictive conditions) as it became more difficult to overcome the barrier represented by the unfavorable energy of activation that accompanies preconditioning (15). This effect of temperature on preconditioning is consistent with the high frequency of cold sensitivity among pyrA arginine auxotrophs. Although examples of the role of small molecules in preconditioning of allosteric enzymes are available (15), evidence for such a role in vivo has been lacking. In this and in a previous paper (5), we have described two kinds of mutations in pyrA, the phenotypes of which are conditionally expressed on the basis of the composition of the intracellular environment. In the first kind, the mutant phenotype is expressed only if the metabolic intermediate N-acetylornithine is present as the enzyme is being synthesized; in the second type, the mutant phenotype is expressed only if the cell lacks OTCase, an associated enzyme. In both cases, the intracellular environment appears to determine the proper quarternary structure of the enzyme. It is reasonable to expect that similar conditionally expressed mutations must occur in genes encoding other multimeric enzymes. The reason one finds this class of mutation so frequently in pyrA must reflect the fact that they express a different phenotype than those mutations that cause complete loss of enzyme function. The distinguishable phenotype reflects the fact that CPSase functioned at a metabolic branch point, and that its regulation differs from that of both pathways to which its product flows. ACKNOWLEDGMENT This work was supported by National Science Foundation grant PCM 76-01752.

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Ingraham. 1969. Control of

carbamylphosphate synthesis in Sabnonella typhimu-

rium. J. Biol. Chem. 244:4033-4038. 2. Abdelal, A., and J. L Ingraham. 1969. Cold sensitivity and other phenotypes resulting from mutation in pyrA gene. J. Biol. Chem. 244:4039-4045. 3. Abdelal, A., and J. L. Ingraham. 1975. Carbamylphosphate synthetase from Salmonella typhimurium. Reg-

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12:385-401. 16. Pierard, A., M. Grenson, N. Glansdorff, and J. M. Wiame. 1973. A comparison of the organization of carbamylphosphate synthesis in Saccharomyces cerevisiae and Escherichia coli, based on genetic and biochemical evidences, p. 493. In S. Prusiner and E. R. Stadtman (ed.), Enzymes of glutamine metabolism, American Chemical Society Symposium. Academic Press Inc., New York. 17. Porter, R. W., M. 0. Modebe, and G. R. Stark. 1969. Aspartate transcarbamylase. Kinetic'studies of the catalytic subunit. J. Biol. Chem. 244:1846-1859. 18. Prescott, L. M., and M. E. Jones. 1969. Modified methods for the determination of carbamylaspartate. Anal. Biochem. 32:408-419. 19. Syvanen, J. M., and J. R. Roth. 1972. Structural genes for ornithine transcarbamylase in Sabnonella typhimurium and Escherichia coli K-12. J. Bacteriol. 110:66-70. 20. Trotta, P. P., L M. Pinkus, R. H. Haschemeyer, and A. Meister. 1974. Reversible dissociation of the monomer of glutamine-dependent carbamyl phosphate synthetase into catalytically active heavy and light subunits. J. Biol. Chem. 249:492-499. 21. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106. 22. Vogel, R. H., W. L Mclelian, A. P. Hirvonen, and H. L Vogel. 1971. The arginine biosynthetic system and its regulation. In H. J. Vogel (ed.), Metabolic pathways, vol. 5. Academic Press Inc., New York. 23. Yan, Y., and M. Demerec. 1965. Genetic analysis of pyrimidine mutants of Salnonella typhimurium. Genetics 52:643-651.