Biochemical Genetics, Vol. 13 Nos. 1/2, 1975

Carbamyl Phosphate Synthesis in Bacillus subtilis Barry Potvin 1'2 and Harry Gooder 1

Received 30 Apr. 1974--Final 17 Oct. 1974

In vitro and "in situ" assays have been developed to test the carbamyl phosphate synthetase (CPSase) activity of a series of pyrimidine-requiring mutants of Bacillus subtilis. The enzyme has been shown to be highly unstable, and was successfully extracted only in the presence of 10% glycerol and 1 mM dithiothreitol (Cleland's reagent). It loses activity rapidly when sonicated or when treated with Iysozyme. Genetic studies, using mutants, indicate that B. subtilis may possess two CPSases. This possibility and its physiological consequences were probed enzymatically. CPSase activity has been shown to undergo inhibition by both uridine triphosphate and dihydroorotate; activation has been demonstrated in response to phosphoribosyl pyrophosphate (PRPP) and (to a lesser extent) ornithine. KEY WORDS: Bacillus subtilis; carbamyl phosphate; pyrimidine; regulation; cotransformation.

INTRODUCTION Carbamyl phosphate synthetase (CPSase, E.C. 2.7.2.5) functions at a pivotal branch point between pyrimidine and arginine biosynthetic pathways. This presents the organism with a complex regulatory problem which may be solved by subjecting the enzyme to control by elements of both pathways or by providing two different enzymes. B.P. was supported by Public Health Service Grant GM-006-85 from the National Institute of General Medical Sciences. 1 Curriculum in Genetics and Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina. 2 Present address: Department of Human Genetics and Development, Columbia College of Physicians and Surgeons, New York, New York. 125 © 1975 Plenum Publishing Corporation, 227 West 17th Street, N e w York, N.Y. 10011. N o part o f thls publication m a y be reproduced, stored in a retrieval system, or transmitted, in any f o r m or by any means, electronic, ~nechanie"al~ photocopying~ microfilming~ recording, or otherwise, without written permission of the publisher,

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The only previous reports concerning the CPSase in wild-type Bacillus subtilis concluded that there was only one such enzyme, as in Eseheriehia coli and Salmonella typhimurium (Reissig et al., 1967; Issaly et al., 1970). The latter report described the regulation at the level of enzyme activity as inhibition by UTP, arginine, uridine, and cytidine 5'-monophosphate (CMP) and concerted repression by growth in arginine and uracil. The present investigation was begun as an attempt to better understand the apparently unique distribution of carbamyl phosphate and in so doing to utilize previously published methods to classify an unknown set of pyrimidine-requiring mutants of B. subtilis according to their CPSase activity. Reliable results were obtained only by using a modification of the assay procedure of Levine and Kretchmer (1971). The preparation of active extracts required the use of stabilization techniques employed specifically for the mammalian enzyme (Tatibana and Shigesada, 1972a). Genetic evidence is presented here which suggests that, unlike the enteric bacteria studied to date, B. subtilis seems to possess two CPSases. Biochemical evidence shows that the regulation of these enzymes more closely resembles that found in yeast and mammalian cells than it does that of the enteric bacteria. MATERIALS AND METHODS Bacterial Strains

Sources of the strains of B. subtilis, S. typhimurium, and E. coli used in this study are listed in Table I. Chemicals

Except where otherwise noted in the text, all pyrimidines, purines, pyrimidine precursors, and amino acids were purchased from Calbiochem. Inorganic compounds and organic solvents were reagent grade and were obtained from either J. T. Baker or Fisher Chemical Co. Potassium penicillin G was from Squibb. The radioactive sodium bicarbonate-C ~4 was originally obtained from New England Nuclear (5 mCi/mmole) and later from Amersham Searle (60 mCi/mmole). Media and Growth Conditions

Media, dilution broth, and growth conditions were as described by Kelleher and Gooder (1973). Unless otherwise stated, distilled water, which was further purified by passage through both charcoal and mixed-bed deionizer columns (Barnstead Still and Sterilizer Co.), was used throughout this work.

127

Carbamyl Phosphate Synthesis in B. subtilis Table I. Bacterial Stock Cultures ~ Strain designation 168MI 168T SB491 W23 ASP12A BK1 SB491U5 H-37 H-59 SB270 SB5 SB305 AU UTT A26U SB319 168TUT

Source

Parent strain

Bacillus subtilis Spizizen Bott (W23 x 168MI) b Carlton Spizizen Spizizen TL (168T + x 168MI) b TL (NTG) Jensen (NTG) Jensen (NTG) Nester Nester Nester Young Young Marmur Nester R0mig and Rothman

168MI 168MI 168MI 168MI SB491 WB746 A139 168MI 168MI 168MI 168MI ? 168MI 168MI 168M!

Nutritional requirements Trp none none none none none Lira none (arg ~) none (ura ~) Ura Trp Ura His Met Ura Arg Ura Trp Thr Ura Ura Met Lira Trp Thy Ura

Other bacterial stocks Escherichia coli B Salmonella typhimurium pyrA 81 pyrA 137

Meynell O'Donovan O'Donovan

none LT-2 LT-2

Arg Ura Ura

"Abbreviations: Arg, arginine; arg ~, arginine sensitivity; His, histidine; Met, methionine; Thr, threonine; Thy, thymine; Trp, tryptophan; Ura, uracil; ura ~, uracil sensitivity; NTG, N-methyl-N'-nitro-N-nitrosoguanidine; TL, this laboratory; ?, unknown. b This notation indicates transformation of strain 168MI- by DNA from strain W23 or 168T ÷.

Mutagenesis and Selection Procedures B. subtilis strains 1 6 8 M I - a n d SB491 (Table I) were the p a r e n t strains in these experiments. E x p o n e n t i a l - p h a s e cultures growing in c o m p l e x m e d i u m were m u t a g e n i z e d b y the direct a d d i t i o n o f 50 #g/ml N - m e t h y l - N ' - n i t r o - N n i t r o s o g u a n i d i n e ( N T G , A l d r i c h Chemicals). The cells were exposed to m u t a g e n for 1 hr, washed, a n d resuspended in c o m p l e t e m e d i u m for overnight growth. D u r i n g the next 4 days, the cells were subjected to daily cycles o f selection b y penicillin, followed b y p h e n o t y p i c expression d u r i n g overnight g r o w t h in liquid m i n i m a l m e d i u m s u p p l e m e n t e d with arginine a n d uracil, a c c o r d i n g to the p r o c e d u r e o f A. S. Issaly (personal c o m m u n i c a t i o n ) .

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Isolation of mutant colonies was accomplished on solid media with the appropriate nutritional supplements. Several flasks were used, but only one mutant of similar phenotype was retained from each flask to insure that each was the result of an independent mutational event.

Growth: Derepressed Condition

Cells were grown in standard minimal medium supplemented with growthlimiting concentrations (5 #g/ml) of uracil or arginine. Adequate aeration was provided by shaking on a variable-speed rotary shaker (Eberbach Corp., model 6140) at 150 rpm. The derepressed cultures were harvested by centrifugation in a Sorvall RC2 at 10,400g for 20 rain using the GSA rotor. After one washing with Spizizen's salts (Spizizen, 1958), the cells were resuspended to 250 mg wet weight/ml of 50 mM tris-HC1 buffer, pH 7.5, for cell disruption or overnight storage in liquid nitrogen. Quick freezing was accomplished in a - 4 5 C ethylene glycol low-temperature bath (Thermovac Industries Corp.). Uracil- and arginine-independent strains were grown in medium that lacked exogenous uracil and/or arginine. All other conditions were as above.

Growth: Repressed Condition

Growth medium used for the repressed condition contained normal amounts (50 #g/ml) of uracil and/or arginine except where otherwise noted in the text. Cultures were harvested routinely at the end of exponential-phase growth as determined by turbidity readings in order to obtain the maximum number of metabolically active cells. All other aspects of the procedure were as stated above.

Methods of Cell Breakage

Lysozyme Treatment To the resuspended cell pellet described above, enough 100K glycerol was added to achieve a final concentration of 20~. Crystalline egg white lysozyme (muramidase, Worthington Biochemical Corp.) was then added (final concentration 200 #g/ml). The culture lysed during the following 45-rain incubation at 37 C. Except where otherwise noted in the text, Brij-35 was then added (final concentration 1~). Dispersion of this lysed cell suspension was completed by a brief period of sonication (15 sec) at 0 C using a Fisher ultrasonic generator. The crude extract was centrifuged in the RC2 (SS34 head) at 15,000g for 15 min for CPSase or at 25,000g for 60 min for the other enzymes°

Carbamyl Phosphate Synthesis in B. subtilis

129

French Pressure Cell The cell suspension was passed through a French pressure cell (American Instrument Co.) at a pressure reading maintained at 20,000 lb and at a temperature of approximately 4 C. The lysed suspension was then centrifuged and dialyzed against the dithiothreitol-glycerol containing buffer as described under the section "Stabilized Extracts." Sonieation Breakage of E. coli and S. typhimurium was accomplished by sonication in an ice bath for 30 sec with 30-sec intervals between treatments. Sonication was continued for a total of 3-5 rain or until breakage was nearly complete as determined by a visible clearing of the suspension. Centrifugation and dialysis were as before. Stabilized Extracts Various compounds were added to the cell suspension prior to breakage in an effort to stabilize the CPSase during extraction. Sucrose-stabilized extracts (SSE) contained 0.25 M sucrose, 1 mM ATP, and 2 mM MgC12 (Tatibana and Ito, 1967). Glycerol-stabilized extracts (GSE) were made by resuspending the cells in 50 mM tris-acetate, pH 7.5, containing 5/zM MnC12, 1.0 mM dithiothreitol (Calbiochem), and 10~o (w/v) glycerol (Tatibana and Shigesada, 1972a). Other stabilization agents tested were 2 mM ornithine, 30% dimethylsulfoxide (DMSO, Mallinckrodt), DMSO plus glycerol in the ratio of 6:1 (Jones, 1971), 0.05 mg/ml phenylmethylsulfonylfluoride (PMSF, Sigma), 3 mM 2-mercaptoethanol, and 1 mM glutathione. In all cases, the agents were added prior to breakage according to the previously described procedure under "French Pressure Cell." Damaged Cell Preparations The cells were resuspended in 50 mM tris-acetate, pH 7.5, containing 5 /tM MnC12 to a concentration of 250 mg wet weight cells per milliliter. To this suspension was added 0.1 ml 1% cetyltrimethylammonium bromide (CETAB, J. T. Baker) and 0.05 ml of 10% sodium desoxycholate (DOC, Fisher Scientific Co.) per milliliter of cells. After shaking for 5 sec, the cells were dialyzed for 4-7 hr against the same tris-acetate and MnC1z buffer. Suspensions handled in this manner will hereafter be referred to as "DCP" (Kaminskas and Magasanik, 1970). Assay Procedures Protein Determinations Protein concentrations of extracts and column fractions were determined

130

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using a slight modification of the technique of Lowry e t al. (1951) with bovine serum albumin as the standard. Carbamyl Phosphate Synthetase Assay

The reaction mixture for the CPSase assay consisted of 0.1 ml of 1 M tris-HC1, pH 7.5, 0.2 ml of a solution of 100 mM K-ATP (P & L Biochemicals), and 140 mM MgC12 (adjusted to pH 7.1 with KOH), 0.1 ml of 100 mM glutamine, 0.1 ml of 55 mM NaHC1403 (Amersham Searle) diluted to 4.0-7.0 × 105 cpm with KHCO3, and 0.1 ml extract in a total volume of 0.85 ml. Incubation was at 37 C for 30 rain. All other aspects of the procedure were as previously described (Levine and Kretchmer, 1971). Appropriate corrections were made by subtracting the activity from both no substrate (no glutamine added) and no enzyme controls. Extracts were made from two different strains of S. typhimurium reported to contain deletions for each of the first two enzymes of the pyrimidine biosynthetic pathway (G. O'Donovan, personal communication). Extracts of wild-type E. eoli B were employed to provide additional controls for each assay. Radioactive determinations were done in a Triton X-100 plus toluenebased scintiilation fluid in a Packard Tricarb liquid scintillation spectrophotometer (model 3320). CPSase activity is expressed as "relative activity" using the activity of wild-type strain BK1 as the standard. Other CPSase assay procedures were employed, including that used by Issaly et al. (1970). All were found to yield poorly reproducible, unsatisfactory results. Further details of these and other methods and modifications used in this work are available (Potvin, 1973). Separation of Enz)~mes

Ammonium Sulfate Fractionation

The carbamyl phosphate synthetase in GSE preparations was partially purified by the method of Issaly et aL (1970). Precipitated protein was collected at 1.5, 2.0, 2.5, and 3.0 M concentrations of ammonium sulfate (ultrapure enzyme grade, Schwarz-Mann). These concentrations are equivalent to 36.6, 48.8, 61.0, and 73.2% saturations, respectively. At each step, the pelleted precipitate was taken up in a small amount (approximately 1.0 ml) of the glycerol-dithiothreitol containing buffer. All fractions were dialyzed against this buffer in the cold for 4-8 hr. Gel Filtration

The gel filtration method of Wiltiams and Davis (1970) was used in an attempt to demonstrate separable CPSase activities. A 37.5 by 1.2-cm bed of Biogel A-1.5 M 100-200 mesh (Bio-Rad) was constructed in a Kontes glass column

Carbamyl Phosphate Synthesis in B. subtilis

131

(50 by 1.2 cm). GSE buffer was used to equilibrate the column and to serve as the eluant. Void volume was determined to be 19.5 ml, and 0.5-ml fractions were collected using a Golden Pup Retriever (Isco). Sucrose Density Gradients The method of Shoal and Jones (1971) was used for sucrose density gradients. GSE buffer was substituted for the tris-sucrose-glycerol-DMSO buffer originally employed. D N A Extraction

A modification of the method of Marmur (1961) as suggested by Dr. Kenneth Bott (personal communication) was used for DNA extraction. Details may be found elsewhere (Potvin, 1973). Growth of Competent Cells

Competent recipient cells were grown according to the procedure of Wilson and Bott (1968) in the laboratory of Dr. Kenneth Bott. Transformation

As suggested by Dr. Bott, a modification of the procedure of Wilson and Bott (1968) was used for transformation. The DNA concentrations were adjusted such that the transformations were being done at or slightly below the saturation level of 1.0/~g/ml (Kelleher and Gooder, 1973). Other aspects of this procedure have been described in detail elsewhere (Kelleher, 1969; Potvin, 1973). Cold shock was strictly avoided during this procedure in order to prevent loss of the potential transformants by thermal stress (Neale and Chapman, 1969). Transformed cultures were held at room temperature with occasional gentle mixing and plated out on selective media within 1 hr following the addition of deoxyribonuclease (DNase). RESULTS Mutant Strains

Attempts to isolate mutants of B. subtilis defective in CPSase activity were only marginally successful. Both strains available in our stocks, "168TUT" and "SB491U5," had defects in other enzymes of the pyrimidine biosynthetic pathway in addition to their lack of CPSase activity. Two other mutants (defective only in CPSase) were obtained from Dr. Roy Jensen. Designated "H-37" and "H-59" by Jensen, the former was sensitive to arginine (inhibi-

132

Potvin and Gooder

tion reversed by uracil) a n d the latter was sensitive to uracil (inhibition reversed by arginine). A n additional 25 pyrimidine m u t a n t s were isolated in this laboratory, a n d 23 others were obtained from other investigators; these have other enzymatic defects (B. Potvin et al., u n p u b l i s h e d data) a n d will be merely m e n t i o n e d here. A d d i t i o n a l i n f o r m a t i o n concerning the strains used in this study will be f o u n d in Table I.

Demonstration of CPSase Activity Activity was initially d e m o n s t r a t e d for CPSase using damaged cell preparations a n d a n assay procedure which converts b o t h c a r b a m y l phosphate-C 14 a n d cyanate-C 14 to hydroxyurea-C 14 (Levine a n d Kretchmer, 1971). The sensitivity of this assay is quite high, with less t h a n 2 % of the total carbamyl phosphate formed escaping as C~40 2. U n d e r the conditions of this assay, a linear increase in counts per m i n u t e incorporated as p r o d u c t was observed between 10 a n d 30 rain. Relative activities of several wild-type a n d m u t a n t strains are f o u n d in Table II.

Table II. Relative Activities for CPSase of Some Wild-Type and Mutant Strains Using In Situ Assay Conditions" Growth conditions: additions to minimal medium Strain Wild type BK1 SB491 ASP12A W23 168MIMutants SB491U5 168TUT H-37 H-59 Salmonella deletion mutants pyrA 81 (CPSase-) pyrB 137 (ATCase-)

Arginine

Uracil

Uracil and arginine

None

1.00 0.73 0.56 0.72 0.48

0.06 -----

0.00 -----

1.03 0.78 0.60 0.83 0.54

0.00 0.00 -0.08

0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00

0.01 0.17 0.01 0.09

---

---

---

0.00 11.15

" Final concentration of both arginine and uracil in the growth medium was 50 pg/ml. One unit of relative activity is approximately equal to 0.750 nmole product formed/min/mg protein at 37 C. The specific radioactivity of the diluted bicarbonate was about 200 cpm/nmole. The CPSase relative activities of the 48 other pyrimidinerequiring mutants of B. subtilis ranged from 0.30 to 50.56; all have defects in other enzymes of the pyrimidine biosynthetic pathway. From B. Potvin et al. (unpublished data).

Carbamyl PhosphateSynthesisin B. subtilis

133

Preparation of Extracts The CPSase was successfully extracted using stabilization techniques which have p r o v e n useful in w o r k i n g with the m a m m a l i a n CPSase. Details are given in M a t e r i a l s a n d M e t h o d s a n d are referred to as " G S E " a n d " S S E . " The best results were achieved using " G S E " p r e p a r a t i o n s . Brief (30-see) sonication resulted in a loss o f activity, as d i d the omission o f glycerol or d i t h i o t h r e i t o l f r o m the e x t r a c t i o n buffer or exposure to lysozyme (Table III). I n o u r hands, all CPSase p r e p a r a t i o n s f r o m B. subtilis are highly unstable, requiring storage in liquid nitrogen. T h e a d d i t i o n o f p r o t c a s e inhibitors such as phenylmethylsulfonylfluoride ( P M S F , Sigma) or other stabilization agents h a d no significant effect on the rate o f d e c a y o f CPSase activity. E n z y m e activity in extracts f r o m b a c t e r i a o t h e r t h a n B. subtilis was m u c h m o r e stable.

Table III. Effects of Some Additions to the Reaction Mixture and Alterations in the Extraction Procedure on CPSase Activity Wild-type strain BK1-DCP Addition to reaction mixture

Concentration (pg/ml)

CPSase relative activity"

None RNase DNase Bovine serum albumin Streptomycin Lysozyme Autoclaved lysozyme Autoclaved BSA

0 200 200 200 200 200 200 200

1.00 1.20 0.96 0.95 0.57 0.13 0.74 0.81

Wild-type strain BK1-GSE Treatment of extract

CPSase relative activity

Control Minus glycerol Minus dithiothreitol Plus sonication

1.00 0.35 0.00 0.00

"One unit of relative activity is approximately equal to 0.805 nmole product formed/rain/rag protein at 37 C. DCP assays contain enough cells (approximately 10 9) to yield a protein concentration of about 1.0mg/reaction vessel. GSE assays contain about 1.5 mg protein/ reaction vessel. The specific radioactivity of the diluted bicarbonate was about 175 cpm/nmole.

134

Potvin and Gooder Characterization of the CPSase

Optimum Harvest Time In order to determine the optimum phase of growth to harvest cells for CPSase assay, samples were removed from a growing wild-type culture, treated by the DCP method, and assayed. Wild-type strains were found to have the highest activity when harvested during late exponential phase (about 5-6 hr after inoculation). 3

Optimum p H and Substrate Combinations Determinations of optimum p H and substrate combinations were made using BK1-DCP and "SSE" preparations as the source of enzyme. The final p H of the reaction mixture was altered by changing the p H of the 1 M trisHC1. Figure 1 presents the effects of varying the p H between 7.0 and 9.0, and resulted in the choice o f p H 7.5 as the standard for further work. Background levels in the controls did not show any significant change over the p H range tested. Concentrations of both A T P and glutamine were varied and levels of CPSase activity tested. Figure 2 shows the results for ATP, while Fig. 3 presents similar data for various glutamine concentrations. Standard values for future experiments were set at 10 pmoles glutamine and 20/~moles K-ATP. Background activity in the control without enzyme showed no apparent changes over the range of concentrations tested. The control without substrate, however, was significantly higher at a K - A T P level of 10 pmoles. 3 It has recently been found that the concentration of excreted uracil in the growth medium of B. subtilis is highest during midexponential phase (leading to a significant repression of the pyrimidine biosynthetic enzymes) and decreases with further growth (Womack, 1973).

I - 30C o Q 0

Q

20C 0 O.

I00

6 -~ o

71o

;5

pH

81o

815

.....

Fig. 1. Response of carbamyl phosphate synthetase activity from B. subtilis wild-type strain BK1 to variations in the final pH of the reaction mixture. DCP assays contained enough cells (approximately 10 9) to yield a protein concentration of about 1.0 rag/reaction vessel. SSE assays had a protein concentration of a!cproximately 1.5 rag/reaction vessel. The specific radioactivity of the diluted bicarbonate was 140 cpm/nmole. Other assay conditions were as 4o described in the text. e, DCP preparation; ©, SSE preparation.

Carbamyl Phosphate Synthesis in B. subtilis

135 400

300 0 r,-

0

Fig. 2. Response to carbamyl phosphate synthetase activity from B. subtilis wild-type strain BK1 to variations in the ATP concentration of the reaction mixture. Protein concentrations were the same as those described for Fig. 1. The specific radioactivity of the diluted bicarbonate was about 130 cpm/nmole. Other assay conditions were as described in the text. The data presented in this figure were used only to determine the optimum ATP concentration for future assays, o, DCP preparation; o, SSE preparation.

200

i

£

.u rnoles K - A T P

400

i-

8 ~°°1 :

°

fl I< ~ 200 13.

Fig. 3. Response of carbamyl phosphate synthetase activity from B. subtilis wild-type strain BK1 to variations in the glutarnine concentration of the reaction mixture. Protein concentrations were the same as those described for Fig. 1. The specific radioactivity of the diluted bicarbonate was about 130 epm/nmole. Other assay conditions were as described in the text. The data presented in this figure were used only to determine the optimum glutamine concentration for future assays, e, DCP preparation; o, SSE preparation.

f_l

_z O2 d

JJrnoles G L U T A M i N E

Glutamine--The Primary Nitrogen Souree The difficulty e n c o u n t e r e d in trying to assay the CPSase in B. subtilis led to the suspicion t h a t the e x t r a c t i o n procedures m i g h t be causing a dissociation o f the e n z y m e into a " r e g u l a t e d synthesis s u b u n i t " and a " g l u t a m i n e utilization

136

Potvin and Gooder

subunit" as had been reported for the E. coli CPSase (Trotta et al., 1971). To test this possibility, 50/~moles NH4C1 was substituted for the 10/~moles glutamine as the nitrogen donor in the reaction mixture under conditions which should inhibit any interfering enzymes (Issaly et aI., 1970). Incorporation of NaHC1403 into product was found to be very low at pH 7.5, increasing slightly at higher pH values. The amount of radioactive product formed when NH4C1 was used never exceeded 25% of the levels observed with fivefold lower concentrations of glutamine. Unsuccessful attempts were also made to recover activity by recombining small amounts of the pelleted materials with the "SSE" supernatant. It was concluded that NH4C1 was utilized much less efficiently than glutamine as a source of nitrogen by the CPSase from B. subtilis. Partial Purification by Ammonium Sulfate Fractionation

Ammonium sulfate fractionation of SSE preparations resulted in an irreversible loss of CPSase activity. Using GSE, it was found that the enzyme precipitated at (NH4)2SO4 concentrations of 1.5-2.5 M. This procedure also produced approximately a fivefold increase in specific activity compared to the original extract. The required presence of 10% glycerol made further purification difficult. Passage through Sephadex G25 was not feasible and dialysis had to be used, even though it was undoubtedly a much rougher treatment for this unstable enzyme. In yeast, DEAE-Sephadex column chromatography destroyed CPSase activity (Lue and Kaplan, 1969). Presence of Two CPSases?

Strain BK1 (Table I) was grown under derepressed, arginine-repressed, and uracil-repressed conditions. For this experiment, the concentration of uracil had to be reduced to 7.5 #g/ml for the uracil-repressed culture in order to retain detectable levels of CPSase activity. Profiles of activity derived from gel filtration of GSE preparations of these cultures are presented in Fig. 4. Similar experiments were performed using extracts of strains H-37 and H-59 (Table I); however, the low activities of these two mutants rendered these results difficult to interpret. These data prompted us to consider the putative presence of more than one CPSase in B. subtilis. Conventional attempts to demonstrate more than one CPSase were also made by sucrose density gradient centrifugation of both strain BK1 derepressed GSE extracts and the ammonium sulfate (1.5-2.5 M) precipitated activity. The former yielded no detectable activity after the 20-hr centrifugation, while with the latter preparation all the remaining activity was recovered as a single peak spread over three 0.5-ml gradient fractions.

Carbamyl Phosphate Synthesis in B. subtilis

137

1200

0 tic o-

I000

800

~:

600

2 ~7

400

r~ (J

200

d 0

6

8 I0 12 14 ELUTION VOLUME (rnl)

16

18

Fig. 4. Elution patterns of carbamyl phosphate synthetase activity from B. subtilis wild-type strain BK1 following growth under arginine-repressed, uracil-repressed, and derepressed conditions (see Materials and Methods). Extracts (GSE) contained 10% (w/v) glycerol and 1 rnM dithiothreitol. These elution patterns were reproducible using independently prepared extracts. The specific radioactivity of the diluted bicarbonate was 160 cpm/nmole. The protein concentration of the extracts before passage through the column was about 20 mg/ml. Each 0.5-ml fraction was assayed for CPSase activity as described in the text. Other details concerning the column and fraction collection may be found in Materials and Methods. e, Derepressed; II, arginine repressed; A, uracil repressed.

Regulation Studies Repression It became obvious during these experiments that uracil and, to a much lesser extent, arginine, when added to growth media in amounts greater than 5 /~g/ml, would lower detectable CPSase activity. Since mutants blocked in each of several pyrimidine interconversion reactions are not yet available in B. subtilis, it is not possible to determine which of the many possible uracilderived products may be directly responsible for the uracil repression. Such experiments done in S. typhirnurium indicated that a cytosine compound was the pyrimidine causing the repression while uracil had no effect (Abd-E1-A1 and Ingraham, 1969a).

Inhibition and Activation A number of pyrimidine pathway intermediates, pyrimidine or purine nucleo-

138

Potvin and Gooder Table IV. Inhibition and Activation of the CPSase from WildType Strain BK1-DCP

Addition to reaction mixture None PRPP Ornithine Acetylglutamate Arginine Arginine Uridine Carbamyl phosphate Dihydroorotate UTP UTP plus arginine UMP IMP Avidin Biotin

Concentration

Relative activity~

-10 pmoles 10 pmoles l0/tmoles 10 pmoles 100/lmoles 100 pmoles 100/~moles 100 pmoles 10/zmoles 10 pmoles each 10 ¢tmoles 10 pmoles 1 unit 10/zmoles

1.00 3.76 2.05 1.54 1.09 0.87 0.72 0.45 0.18 0.18 0.0l 0.58 1.05 0.10 0.84

, One unit of relative activity is approximately equal to 0.735 nmole product formed/min/mg protein at 37 C. DCP assays contain enough cells (approximately 109) to yield a protein concentration of about 1.0 mg/reaction vessel. The specific radioactivity of the diluted bicarbonate was about 130 cpm/ nmole. Note that these assays were done on damaged cell preparations of wildtype B. subtilis and that observed activations and inhibitions could be affecting either one or both of the two proposed CPSase enzymes.

sides or nucleotides, a m i n o acids, a n d cofactors or inhibitors were a d d e d to B K 1 - D C P - c o n t a i n i n g reaction mixtures. In Table IV are listed the concentrations used a n d the effects p r o d u c e d . M o s t significant are the observed activation b y P R P P a n d ornithine a n d the inhibition by avidin, 4 U T P (with or w i t h o u t arginine), a n d possibly d i h y d r o o r o t a t e . I n later work, nearly identical results were o b t a i n e d when B K 1 - G S E was e m p l o y e d as the source o f enzyme. Genetic Evidence for Two CPSases

D N A was extracted f r o m strains H-37 a n d H-59 for use in t r a n s f o r m a t i o n experiments which utilized strains SB5 a n d SB319 (Table I) as the recipients. The a r g s locus o f H-37 was f o u n d to c o t r a n s f o r m with the SB5 u r a - a n d the SB319 u r a - loci 22.5% a n d 32.1% o f the time, respectively. N o such cot r a n s f o r m a t i o n with either recipient was observed for the ura ~ locus o f H-59. 4 Avidin was used to test possible biotin dependence, which was once reported to exist for the E. coli CPSase (Wellner et a l , 1968).

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While these data are not sufficient to establish a map position for the H-37 mutation, they do provide strong evidence that a pyrimidine-specific CPSase locus is in the same area as all the previously established ura- mutations (Young and Wilson, 1972). Furthermore, the lack of cotransformation of the mutation in strain H-59 with any of the ura- markers provides evidence that B. subtilis may have two separate regions of D N A which encode for two CPSase functions. It is difficult, if not impossible, for us to reconcile these reproducible genetic data with the idea of two different subunits of one enzyme, as has been suggested for the CPSase of E. coli (Trotta et al., 1974; Anderson and Marvin, 1970) and S. typhimurium (Abd-E1-A1 and Ingraham, 1969b). We prefer the simpler and currently more logical explanation that B. subtilis may possess two CPSases. DISCUSSION Attempts to assay CPSase using techniques described for other bacteria were unsuccessful. The procedure which had previously been used by Issaly et al. (1970) could not be repeated. The major source of difficulty was assumed to be the extreme lability of the enzyme. This problem was ameliorated somewhat by using "in situ" assay conditions. Cells which had been rendered permeable to substrates by careful treatment with toluene or detergents are believed to approximate the physiological conditions of the enzyme with respect to concentration and interactions with other cellular proteins (Reeves and Sols, 1973). Unstable enzymes in B. subtilis and other organisms are frequently assayed this way (Kaminskas and Magasanik, 1970). It was readily possible for the first time, using this procedure, to determine whether or not the mutants had CPSase activity and to relate the levels to those found in wildtype cells. All the strains assayed for this enzyme were grown under uracilderepressed, arginine-repressed conditions to avoid possible false positive results which might stem from an arginine-specific enzyme. In order to conduct experiments to determine whether there was one CPSase or more, it was necessary to extract the enzyme(s) using those methods employed to stabilize the labile CPSase of mammalian tissues. The most important aspects of the extraction procedure are the presence of glycerol and dithiothreitol in the buffer and the use of the French pressure cell instead of lysozyme or sonication to break the cells. The lysozyme sensitivity of the CPSase from B. subtilis suggests that the enzyme may be associated with membraneous or particulate elements of the cell or that it may be an acidic protein that would complex with basic proteins such as lysozyme or avidin. Previous studies of the CPSase activity in B. subtilis may well have been complicated by one of these problems. When using crude extracts, Issaly used pH 7.5 and glutamine as the

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nitrogen donor to assay only CPSase activity. It seems unlikely that either of the two interfering enzymes reported by Reissig et al. (1967)--acetyl phosphokinase and carbamate kinase--are active here, since they both utilize ammonia as a nitrogen source. While unsuccessful attempts were made to demonstrate a glutamine-utilization subunit such as has been reported for the CPSase of E. coli (Trotta et al., 1971), a B. subtilis mutant has apparently been isolated which seems to lack the ability to use glutamine as the nitrogen donor in a number of different reactions. This mutation maps between cysA and pab, quite far from the pyrimidine locus (J. Kane, personal communication). When we tested this strain, it had a reduced activity for the glutaminedependent CPSase. If B. subtilis possessed only one CPSase, as reported by Issaly et al. (1970), one would expect to frequently observe a requirement for both arginine and uracil in CPSase mutants. The dual requirement should also revert at a frequency expected for a single-point mutation. Our results do not conform to these expectations. The patterns of activity observed after gel filtration of glycerol-stabilized extracts, although not positive proof, were highly suggestive of more than one CPSase in B. subtilis. This hypothesis found further support when two independently induced mutants (H-37 and H-59) were found to have precisely the nutritional and biochemical properties expected of pyrimidine-specific CPSase (CPSas%yr) and arginine-specific CPSase (CPSaseArg) defects. Genetic studies of these strains confirmed that the CPSas%y r mutation cotransformed with all other pyrimidine loci, while the CPSaseArg marker did not. On the basis of the recombination index data for the multiple mutants 168TUT and SB491U5, it is likely that pyrA 5 will eventually be positioned at the far left side of the fine-structure map of the pyrimidine locus as it appears in Young and Wilson (1972). Similar results concerning the mapping of CPSasepy~ mutations of B. subtilis have been obtained by J. Rebello, K. Foltermann, and G. O'Donovan (personal communication). Another strain which is probably mutated for CPSaseA~g and display suracil sensitivity ("ura s'') has been found to be closely linked by transformation to argC (Hoch and Mathews, 1972). Among the 50 Pyr- mutant strains tested in the present study, only two seem to have defects in CPSase activity, and these both have additional enzymatic defects. Strain SB491U5 (Table I) lacks activity for CPSas%y~, DHOase, and DHO-DHase, while strain 168TUT (Table I) is deficient in CPSas%y~, ATCase, and DHOase (B. Potvin et al., unpublished data). 6 It 5 The use of "pyrA" should not be considered to indicate the dual requirement for arginine and uracil observed in E. eoli and S. typhirnuriurn. 6 These two mutants were isolated as single-step mutants, but the possibility of multiple mutations has not been ruled out. A complete report on the enzymatic defects of the 50 pyrimidine mutants of B. subtilis mentioned here will appear in a separate publication.

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appears that a mutation affecting only the CPSasepy r is not sufficient to produce a nutritional requirement for pyrimidines. Strains with this defect would still be able to grow on minimal medium (presumably utilizing carbamyl phosphate produced by CPSaseArg) and would become sensitive to arginine. This would explain why no mutants with only a CPSas%yr defect (pyrA) were isolated in this study since such strains are selected against by the penicillin treatment. In future work, it will be necessary to include arginine in the selection medium to inhibit the growth of pyrA mutants during penicillin selection. The regulation of the B. subtilis CPSase was studied under both "in situ" and in vitro assay conditions. The results were very similar or identical in both systems. Unless otherwise indicated, CPSase refers to both CPSas%yr and CPSas%~g. In many ways, the CPSase of B. subtilis seems to be more similar to that described for Neurospora, yeast, and mammalian cells than to the enzyme found in most other bacteria. Instead of being feedbackinhibited by UMP, it is strongly inhibited by UTP. As has already been found for mammalian cells (Tatibana and Shigesada, 1972b), PRPP exerts a strong activating effect on the B. subtilis CPSase. In Neurospora, there seem to be two enzymes, both utilizing glutamine as the nitrogen donor, one for the arginine and one for the pyrimidine biosynthetic pathway (Davis, 1972). Only the CPSas%y~ is inhibited by UTP, although the enzyme is reported to be in a complex with ATCase (Jones, 1972; Williams et al., 1971). The situation in B. subtilis seems to be very similar, including the association of CPSas%yr with ATCase in a multienzyme complex (B. Potvin et al., unpublished data). This observation, together with the possibility of two CPSases, has obvious implications with regard to channeling of carbamyl phosphate and should be compared to the situation in yeast and Neurospora (Davis, 1972). One property that the B. subtilis CPSase seems to share with other bacteria is its activation by ornithine. It is not known at this time whether ornithine affects both (assuming there are two) or only one of the CPSase enzymes. Previous reports indicate that B. subtilis and S.faecaIis are the only bacteria known to have an ATCase that is not feedback-inhibited (Chang and Jones, 1974; Bethell and Jones, 1969; Neumann and Jones, 1964). It will be interesting to determine whether the CPSase of S. faecalis is regulated in a manner similar to that of B. subtilis and to inquire as to how widespread the phenomenon of CPSase activation by PRPP seems to be. ACKNOWLEDGMENTS We wish to thank Dr. Kenneth Bott for making his equipment and expertise available to us and Dr. Gerald O'Donovan and Dr. Herbert Rosenkranz for their critical reading of this manuscript.

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REFERENCES

Abd-E1-AI, A., and Ingraham, J. L. (1969a). Control of carbamyl phosphate synthesis in Salmonella typhimurium. J. Biol. Chem. 244(15):4033. Abd-El AI, A., and Ingraham, J. L. (1969b). Cold sensitivity and other phenotypes resulting from mutation in pyrA gene. J. Biol. Chem. 244(15):4039. Anderson, P. M., and Marvin, S. V. (1970). Effect of allosteric effectors and adenosine triphosphate on the aggregation and rate of inhibition by N-ethylmaleimide of carbamyl phosphate synthetase of Escherichia coll. Biochemistry 90):171. Bethell, M., and Jones, M. E. (1969). Molecular size and feedback regulation characteristics of bacterial aspartate transcarbamylase. Arch. Biochem. Biophys. 134:352. Chang, T.-Y., and Jones, M. E. (1974). Aspartate transcarbamylase from Streptococcus faecalis: Purification, properties and nature of an allosteric activator site. Biochemistry 13(4) :629. Davis, R. H. (1972). Metabolite distribution in cells. Science 178:835. Hoch, J., and Mathews, J. (1972). Genetic studies in Bacillus subtilis. In Halvorson, H., Hanson, R., and Campbell, L. (eds.), Spores, Vol. V, American Society for Microbiology, Washington, D.C., pp. 113-116. Issaly, I. M., Issaly, A. S., and Reissig, J. L. (1970). Carbamyl phosphate biosynthesis in Bacillus subtilis. Biochim. Biophys. Aeta 198:482. Jones, M. E. (1971). Regulation of pyrimidine and arginine biosynthesis in mammals. Advan. Enzyme Regul. 9:19. Jones, M. E. (1972). Regulation of uridylic acid biosynthesis in eukaryotic cells. Current Topics Cell. Regul. 6:227. Kaminskas, E., and Magasanik, B. (1970). Sequential synthesis of histidine-degrading enzymes in Bacillus subtilis. J. Biol. Chem. 245(14):3549. Kelleher, R. (1969). Genetic, physiological and biochemical studies in Bacillus subtilis. Ph.D. dissertation, University of North Carolina at Chapel Hill. Kelleher, R., and Gooder, H. (1973). Genetics and biochemistry of pyrimidine biosynthesis in Bacillus subtilis: Linkage between mutations resulting in a requirement for uracil. J. Bacteriol. 116(2):577. Levine, R. L., and Kretchmer, N. (1971). Conversion of carbamyl phosphate to hydroxyurea~An assay for carbamyl phosphate synthetase. Anal. Biochem. 42:324. Lowry, D. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. Lue, P. F., and Kaplan, J. G. (1969). The aspartate transcarbamylase and carbamyl phosphate synthetase of yeast: A multifunctional enzyme complex. Biochem. Biophys. Res. Commun. 34(4):426. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208. Neale, E. K., and Chapman, G. B. (1969). Effect of thermal stress on the growth and fine structure of Bacillus subtilis. Bacteriol. Proc. 69:39. Neumann, J., and Jones, M. E. (1964). End-product inhibition of aspartate transcarbamylase in various species. Arch. Biochem. Biophys. 104:438. Potvin, B. (1973). The de novo pyrimidine biosynthetic pathway of Bacillus subtilis. Ph.D. dissertation, University of North Carolina at Chapel Hill. Reeves, R., and Sols, A. (1973). Regulation of Escherichia coli phosphofructokinase in situ. Biochem. Biophys. Res. Commun. 50(2):459. Reissig, J. L., Issaly, A. S., and Issaly, I. M. (1967). Arginine-pyrimidine pathways in microorganisms. Natl. Cancer Inst. Monogr. 27:259. Shoal', W. T., and Jones, M. E. (1971). Initial steps in pyrimidine synthesis in Ehrlich ascites carcinoma. Biochem. Biophys. Res. Commun. 45(3):796. Spizizen, J. (1958). Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonuclease. Proc. Natl. Acad. Sci. 44:1072.

Carbamyl Phosphate Synthesis in B. subtilis

143

Tatibana, M., and Ito, K. (1967). Carbamyl phosphate synthetase of the hematopoietic mouse spleen and the control of pyrimidine biosynthesis. Bioehem. Biophys. Res. Commun. 26(2):221. Tatibana, M., and Shigesada, K. (1972a). Activation by 5-phosphoribosyl 1-pyrophosphate of glutamine-dependent carbamyl phosphate synthetase from mouse spleen. Biochem. Biophys. Res. Commun. 46(2):491. Tatibana, M., and Shigesada, K. (1972b). Control of pyrimidine biosynthesis in mammalian tissues. V. Regulation of glutamine dependent earbamyl phosphate synthetase: Activation of 5-phosphoribosyl 1-pyrophosphate and inhibition by uridine triphosphate. J. Biochem. 72(3):549. Trotta, P., Burt, M., Haschemeyer, R., and Meister, A. (1971). Reversible dissociation of carbamyl phosphate synthetase into a regulated synthesis subunit and a subunit required for glutamine utilization. Proc. Natl. Acad. Sci. 68(10):2599. Trotta, P., Estis, L., Meister, A., and Haschemeyer, R. (1974). Self-associationand allosteric properties of glutamine-dependent carbamyl phosphate synthetase: Reversible dissociation to monomeric species. J. Biol. Chem. 249(2):482. Wellner, V., Santos, J., and Meister, A. (1968). Carbamyl phosphate synthetase A biotin enzyme. Biochemistry 7(8) :2848. Williams, L. G., and Davis, R. H. (1970). Pyrimidine-specificcarbamyl phosphate synthetase in Neurospora crassa. J. Bacteriol. 103(2):335. Williams, L. G., Bernhardt, S. A., and Davis, R. H. (1971). Evidence for two discrete carbamyl phosphate pools in Neurospora. J. Biol. Chem. 246(4):973. Wilson, G. A., and Bott, K. F. (1968). Nutritional factors influencing the development of competence in the Bacillus subtilis transformation system. J. Bacteriol. 95:1439. Womack, J. (1973). Pyrimidine overproduction in lower organisms. Ph.D. dissertation, Texas A&M University. Young, F. E., and Wilson, G. A. (1972). Genetics of Bacillus subtilis and other grampositive sporulating bacilli. In Halvorson, H. O., Hanson, R., and Campbell, L. L. (eds.), Spores, Vol. V, American Society for Microbiology, Washington, D.C.