JOURNAL OF BACTERIOLOGY, August 1975, p. 604-615 Copyright © 1975 American Society for Microbiology

Vol. 123, No. 2 Printed in U.S.A.

Pyrimidine Biosynthetic Pathway of Bacillus subtilis BARRY W. POTVIN,1* RAYMOND J. KELLEHER, JR.,2 AND HARRY GOODER Curriculum in Genetics and Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 30 April 1975

Biochemical and genetic data were obtained from a series of 51 Pyr- strains of Bacillus subtilis. The observed enzymatic deficiencies allowed the mutants to be placed into 12 classes, some of which represent defects in more than one of the six known pyrimidine biosynthetic enzymes. Mapping analysis by transformation has shown that all the Pyr- mutations are located in a single small area of the B. subtilis genome. A correlation of the biochemical defects and the genetic data has been made. Those mutations conferring similar enzymatic deficiencies were found to be contiguous on the B. subtilis map. Regulatory aspects of the pyrimidine pathway have also been investigated and are compared to previously reported results from other organisms. Evidence is presented which bears upon the possible physical association of the first three enzymes and the association of at least some of the enzymes of this pathway with particulate elements of the cell. A model for the organization of the enzymes is presented with dihydroorotate dehydrogenase as the central enzyme in a proposed aggregate. In previous publications (23, 34) we have presented genetic evidence for a tightly linked group of mutations affecting the pyrimidine biosynthetic pathway of' Bacillus subtilis and have described some biochemical and genetic aspects of the first enzyme of' that pathway, carbamyl phosphate synthetase (CPSase, EC 2.7.2.5). These studies of' the pyrimidine biosynthetic pathway (Fig. 1) in B. subtilis, a gram-positive rod, were undertaken to test comparative regulation between organisms. B. subtilis offers the advantages of' its ability to grow on a welldefined minimal medium and the availability of' previously established methods for genetic analysis (2). The biochemical properties of' B. subtilis have revealed that it is not only quite different from Escherichia coli but also from many other bacteria. A major site of' regulation in the pyrimidine pathway of many bacteria seems to be aspartate transcarbamylase (ATCase, while in some Pseudomonas and Saccharomyces uridine-5'-triphosphate (UTP) is phate (CTP) is the feedback inhibitor of ATCase; while in some Pseudomonas and Saccharomyces, uridine-5'-triphosphate (UTP) is the negative effector. Nucleotides do not have any significant effect on the ATCase from Neurospora or mammalian cells (7, 8, 14). The

ATCase of B. subtilis is similar to the enzyme found in this latter group, being unaffected by various pyrimidine and purine nucleotides up to concentrations of 1 mM (6, 35). It has also been found that the B. subtilis ATCase undergoes a rapid, energy-dependent inactivation in stationary-phase cells just prior to sporulation (9, 40). Existing data on the regulation of the activities of the pyrimidine biosynthetic enzymes in B. subtilis afforded the conclusion that the control of the pathway is the same as in other microorganisms; however, dihydroorotase (DHOase, EC 3.5.2.3) and carbamyl phosphate synthetase (CPSase, EC 2.7.2.5) were not s.tudied (L. J. Rebello and G. A. O'Donovan, Tex. J. Sci. 24:117, 1972). In the pyrimidine pathways of Saccharomyces and Neurospora a complex composed of' CPSase and ATCase has been observed (18). Mammalian cells have been reported to possess complexes of CPSaseATCase-DHOase and orotidine-5'-monophosphate pyrophosphorylase (OMP-PPase, EC 2.4.2.10)-orotidine-5'-monophosphate decarboxylase (OMP-DCase, EC 4.1.1.23) (1, 14, 19, 22, 37, 38; Fed. Proc. 31:473, 1972). In Serratia marcescens, a complex consisting of DHOase, OMP-PPase, and OMP-DCase has been found (J. Wild and W. L. Belser, Tex. J. Sci. 24:124, 1972; J. Wild, Ph.D. dissertation, University of California, Riverside, 1972). Evidence for the existence of a similar multi-enzyme complex in B. subtilis as originally suggested by R. J. Kelleher (Ph.D. dissertation, University of

'Present address: Department of Human Genetics and Development, Columbia College of Physicians and Surgeons, 630 W. 168th St., New York, N.Y. 10032. 2Present address: Salk Institute, P.O. Box 1809, San Diego, Calif. 92112. 604

605

PYRIMIDINE BIOSYNTHETIC PATHWAY OF B. SUBTILIS

VOL. 123, 1975 HOOC

CH2 .olCH

H2N M2

MO

N"COOH

ATCose

L-Asportic acid

0

N., \ C~ /° NM72 H2

ATP GLUTAMINE HCOj

CPS -1. PYIRA

HN

DHOos

CM

0 0 O00 "N-'N.oCOOH H2N-COPO3H2 Corbomyl-Lcarbomyl

phosphate

lCH2

DHO-DH

~CH

PYR

-0CIN.

PYR C

N

0

"CHC00H

L-Dihydroorotkc

Asportic ocid

ocid 0

11

sCMN

?I

H

OIMP-IrPna

° NCH

HN

-

OT

1

H

PRPP

MHN'

UTP NJCLEIC CTP AC IDS

NC

C

PYR F

N

ribo4-5'-P Undlne-5'-Phosphate

0

Orotic

acid

C

CCOOH

PYR E

rib-5'-P

Orotidne-5- Phosphate

FIG. 1. The de novo pyrimidine biosynthetic pathway.

North Carolina, Chapel Hill, 1969) is presented in this paper. Genetic data on pyrimidine-requiring mutants of B. subtilis obtained in this and previous studies indicated that all known Pyr- mutations were linked (10, 23, 42). This is unique and unlike the situation for any other microorganisms on which genetic data have been published (E. coli [39]; S. typhimurium [36]; Klebsiella pneumoniae [28]; Pseudomonas aeruginosa [16]; Saccharomyces cerevisiae [24]; Streptomyces coelicolor [15 ]; Diplococcus pneumoniae [29]; Coprinus radiatus [12]; Neurospora crassa [3 ]). 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 1. Mutagenesis and selection procedures. Nitrosoguanidine mutagenesis and penicillin selection techniques were as previously described (34). Although the selection technique was also designed to isolate mutants with a dual requirement for both arginine and uracil ("AU"), none was found among the approximately 14,000 colonies checked. The Pyrmutants that were isolated are most probably the result of single-point mutations because of the observed spontaneous reversion frequencies which were usually in the range of 10-7 to 10-g. Chemicals, media, and growth conditions. The sources of chemicals, preparation of media, and growth conditions were described in previous publications (23, 34). All mutant strains were grown under derepressed conditions (the incubation of cultures for 2 h after growth ceased in a defined minimal medium with a limiting concentration of 5 lAg of uracil per ml [34]). Strain BKl was also grown under argininerepressed (50 ug of arginine per ml) or uracil-repressed (50 IAg of uracil per ml) conditions and in the presence of 5 gg of uracil per ml. The other wild-type strains12A, 168MI-, W23, and SB491-were grown in the absence of added uracil or arginine. Growth on intermediates. Attempts were made to

obtain growth of uracil-requiring mutants on three of the intermediates in the pathway. Liquid minimal medium supplemented with 300 Mg of L-dihydroorotate per ml, with 350 Mg of carbamyl aspartate per ml, with 120 sg of orotic acid per ml, or with 50 Mg of uracil per ml was used for this purpose. Unsupplemented minimal medium was used as a control. Growth was monitored by using a calibrated Klett colorimeter with a no. 54 filter. Experiments were also conducted on solid minimal medium supplemented as above. Preparation of extracts. Methods used to prepare extracts have been described previously (34). Glycerol-stabilized extracts contained 10% (wt/vol) glycerol and 1 mM dithiothreitol. Cell extracts prepared by lysozyme treatment with 1%) the addition of BRIJ-35 (final concentration were assayed for enzyme activities with the exception of CPSase activity, which was usually determined with "Damaged Cell Preparations" (34). In some cases noncentrifuged preparations were used to confirm the results found in the 25,000 x g (1 h) extracts. Assay procedures. (i) Enzyme assays. In addition to the usual no-substrate and no-enzyme controls, these assays were also tested with extracts made from strains of S. typhimurium reputed to contain individual deletions for each of the six enzymes of the pyrimidine biosynthetic pathway (41; O'Donovan, personal communication). Extracts of E. coli B were used to provide an additional control for each assay. All enzyme assays were performed at 30 C with the exception of CPSase, which was incubated at 37 C. (ii) CPSase. This enzyme was assayed by the method of Potvin and Gooder (34). (iii) ATCase. This assay was made according to the procedure of Neumann and Jones (30) with minor modifications. Dilithium carbamyl phosphate was obtained from Sigma Chemical Co. and required no further purification. The amount of product formed in this reaction was determined with the method of Prescott and Jones (35). (iv) DHOase. This enzyme was assayed in the reverse direction by using a slight modification of the method of Beckwith et al. (4). The amount of product was determined in the same way as for ATCase. =

606

J. BACTERIOL.

POTVIN, KELLEHER, AND GOODER TABLE 1. Bacillus subtilis stock culturesa Strain

designation 168MI 168T+ SB491 W23 ASP12A BK1 168MIU1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 SB491U1 SB491U2, 3, 4, 5,c 6, 7, 8 168SU-1, 2, 3, 5c, 7c, 8c H-37 H-59 SB270 SB5 BD71 MB45 MB46 MB102 MB106 MB169 SB305 AU UTT A26Uc SB319c SB8c BR72 27-6 WB577 17A-42 168TUTC 168THY - U G18

GSY289 GSY318 Other bacterial stocks E. coli B S. typhimurium pyrA81 pyrB137 pyrC66 pyrD67 pyrE125 pyrF146

Source jSuc r Spizizen Bott (W23 x 168MW' Carlton Spizizen Spizizen TL(168T+ x 168MW)5 TL (NTG)

TL (NTG) TL (NTG) Spizizen (EMS) Jensen (NTG) Jensen (NTG) Nester Nester Dubnau Marmur Marmur Marmur Marmur Marmur Nester Young Young Marmur Nester Nester Young Greer (OHNH2) Nester (UV) Greer (HNO2) Romig (UV) Bresler (Thy starv) Green Anagnostopoulos Anagnostopoulos

Parent strain

168MI 168MI 168MI 168MI 168MI SB491 SB491 168MI WB746 A139 168MI 168MI SB3 168MI 168MI 168MI 168MI 168MI 168MI 168MI 9

168MI 168MI 168MI 168MI 168MI 168MI 168MI 168MI 168MI 168MI 168MI 168MI

Meynell O'Donovan O'Donovan O'Donovan O'Donovan O'Donovan O'Donovan

j

Phenotype nutritional requirements Trp None None None None None Trp Ura Trp Ura Ura Trp Ura None (Arg") None (Ural) Ura Trp Ura His Trp Ura His Trp Ura His Trp Ura His Leu Thy Pur Ura Met Ura Trp Ade Ura Met Ura Arg Ura Trp Thr Ura Ura Met Ura Thr Ura Trp Ura Ura Ura Ura Trp Thy Ura Thy Ura Trp Ura Trp Ura Trp Ura None

LT-2 LT-2 LT-2 LT-2 LT-2 LT-2

Arg Ura Ura Ura Ura Ura Ura

aAbbreviations used: TL, this laboratory; Ade, adenine; Arg, arginine; Arg8, arginine sensitivity; EMS, ethylmethane sulfonate; His, histidine; OHNH2, hydroxylamine; Leu, leucine; Met, methionine; HNO2, nitrous acid; NTG, N-methyl-N'-nitro-N-nitrosoguanidine; Pur, purines; Thr, threonine; Thy, thymine; Trp, tryptophan; Thy starv, thymine starvation; Ura, uracil; Urag, uracil sensitivity; UV, ultraviolet light; and ?, unknown. This notation indicates transformation of strain 168MI - by deoxyribonucleic acid from either strain W23 or 168T+. c These strains were never observed to revert.

(v) Dihydroorotate dehydrogenase (DHO-DHase, EC 1.3.3.1). The modification described by O'Donovan and Gerhart (31) of the procedure of Beckwith et al. (4) was adopted for this assay. (vi) OMP-PPase. This assay was modified from

that of Beckwith et al. (4). The reaction mixture in total volume of 1.2 ml contained 100 ,mol of tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.5, 0.25 gmol of orotic acid, 2.5 ,umol of magnesium chloride, and 0.5 ,umol of tetra sodium 5'-phos-

PYRIMIDINE BIOSYNTHETIC PATHWAY OF B. SUBTILIS

VOL. 123, 1975

phoribosylpyrophosphate (PRPP, Calbiochem). Conversion of orotic acid to orotidine-5'-monophosphate (OMP) was monitored spectrophotometrically with a Gilford 2000 recording spectrophotometer as a function of the decrease in absorbance at 295 nm. To a silica cuvette (1-cm light path, 1.2-ml working volume), buffer, orotic acid, magnesium chloride, an appropriate volume of distilled water, and 0.1 ml of extract were added. The optical density at 295 nm was monitored to determine if any endogenous pyrophosphorylase activity was present. The reaction was initiated by the addition of PRPP. The absorbance change during the initial linear portion of the reaction curve was determined. An optical density decrease of 3.67 units corresponds to the conversion of 1 Mmol of orotic acid (4). (vii) OMP-DCase. The technique used for this assay was a modification of the method of Beckwith et al. (4). The reaction mixture contained 100 smol of tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.5, 0.275 Mimol of OMP, 2.5 gmol of magnesium chloride, 0.1 ml of extract, and enough H2O to make a total volume of 1.0 ml in a silica cuvette (working capacity 1.2 ml, path length 1.0 cm). The reaction was initiated by the addition of OMP. The resulting decrease in absorbance at 285 nm was monitored on a Gilson 2000 recording spectrophotometer which had been set at zero absorbance by using water. A decrease of 1.38 units corresponds to 1 Mmol of substrate decarboxylated. (viii) Catalase. Beef liver catalase (molecular weight = 247,000, Worthington Biochemicals) was used as a marker enzyme in sucrose density gradients. It was assayed by measuring the disappearance of hydrogen peroxide spectrophotometrically at 240 nm

(5). (ix) Protein determinations. Protein concentrations of extracts and column fractions were determined with a slight modification of the technique of Lowry et al. (26) with bovine serum albumin as the standard. Separation of enzymes. Ammonium sulfate fractionation, gel filtration, and sucrose density gradient centrifugation were as previously described (34). Transformation and construction of linkage map. Deoxyribonucleic acid extraction, growth of competent recipient cells, and transformation were as previously described (23, 34). The original linkage map was confirmed and expanded by the same methods used in earlier work (23).

RESULTS

Response to intermediates. Growth to normal stationary-phase levels was obtained in liquid minimal medium supplemented with orotate using strains SB319, BR72, 17A-42, WB577, and 168TUT (Table 1). Dihydroorotate or carbamyl aspartate usually would not support growth. In all cases no growth had occurred in unsupplemented minimal medium by the time growth was recorded in the presence of the intermediate. All strains tested continued to

607

display their original uracil requirement after growth on an intermediate. Preparation of extracts. It has previously been reported in other microorganisms that DHO-DHase seems to be particle-bound (20, 21). Assays for this enzyme were, therefore, performed with crude, noncentrifuged extracts. In our laboratory no difficulty was encountered in assaying DHO-DHase in B. subtilis by using lysozyme extracts that had been centrifuged at 25,000 x g for 30 min. Centrifugation at 100.000 x g for 4 h, however, destroyed DHO-DHase activity when lysozyme extracts had not been treated with the nonionic detergent BRIJ-35. If BRIJ-35 was used, the activity was 10-fold higher in the 100,000 x g pellet than in the supernatant fluid. It was also found that centrifugation at 25,000 x g for 60 min instead of 30 min significantly prolonged the linear activity observed in OMP-PPase assays. If the sedimented material was remixed with the supernatant fluid, the reduced period of linearity was observed once again. Biochemical characterization of the mutants. The levels of activity present in wild-type strains of B. subtilis are presented in Table 2, while similar data for the various enzymes in the mutant strains are presented in Table 3. Enzymatic deficiencies are reproducibly observed and higher activities could not be demonstrated when the amounts of substrate or extract were increased. Extracts from mutants with enzymatic defects do not inhibit the corresponding activity in extracts of wild-type strain BK1. Several strains (BD71, MB45, MB46, MB106, MB169) have the same uracil mutation as strain SB5 (J. Marmur, personal communication). These were included in this study because of a previous report that strain SB5 was deficient in ATCase activity (27). The mutants are grouped in classes in Table 4 according to their enzymatic defects. Attempts to determine the biochemical nature of pyrX mutants. The identity of the biochemical lesion in pyrX mutants proved to be elusive. Earlier work in this laboratory had demonstrated activity for the last five enzymes of the pyrimidine pathway (ATCase through OMP-DCase) and implied that such mutants might lack a uracil-specific CPSase activity. The initial efforts to develop a CPSase assay (34) were aimed at testing this hypothesis. All pyrX mutants had CPSase activity when grown under arginine-repressed conditions (Table 3). In two strains, UTT and A26U (Table 3), CPSase was very low and difficult to detect. In addition, thiamine and citrulline were tested to determine if the pyrimidine requirement of

608

POTVIN, KELLEHER, AND GOODER

J. BACTERIOL.

TABLE 2. Enzymatic activities of some wild-type strains Enzyme activity

Strain

168MISB491 W23 ASP12A BK1 BK1J(U)d

BK1-URAe

CPSasea

ATCaseb

DHOaseb

DHO-DHasee

OMP-PPaseb

OMP-DCaseb

0.48 0.73 0.72 0.56 1.00 0.00 0.00

11.13 17.86 17.88 10.30 23.30 0.00 0.00

4.40 3.04 4.98 3.57 2.01 0.54 0.00

0.32 0.52 1.79 0.91 0.41 0.13 0.00

0.56 0.71 1.19 0.65 1.49 0.42 0.58

8.22 9.45 6.34 13.80 16.51 3.40 0.00

J

aRelative activity at 37 C, based on BK1, all strains arginine-repressed. One unit of relative activity is approximately equal to 0.750 nmol of product formed/min per mg of protein. The specific radioactivity of the diluted bicarbonate was about 200 counts/min per nmol. b The specific activities of ATCase, DHOase, OMP-PPase, and OMP-DCase are expressed as nanomoles of product formed or substrate transformed per minute per milligram of protein at 30 C. One unit of DHO-DHase specific activity is that causing a change of 1.0 optical density unit at 480 nm per mg of protein at 30 C in 20 min. d BK1 grown in presence of 5 ,g of uracil per ml. eBK1 grown in presence of 50 tg of uracil per ml. c

these strains was a secondary effect of an enzymatic defect in another pathway, and it was found that neither compound would support the growth of pyrX mutants. Other possible explanations include a deficiency in a pyr-

imidine-specific phosphoribosylpyrophosphate synthetase (PRPP synthetase, EC 2.7.6.1). Excretion of orotate. It was noted that after prolonged storage of stock cultures on tryptose blood agar plates (Difco) many of the pyrX mutants accumulated deposits of a white crystalline material. This substance was identified as orotate on the basis of its absorption spectrum and its ability to serve as the substrate for purified yeast OMP-PPase (Sigma). This phenomenon was observed in strains SB5, UTT, A26U, AU, BD71, 168MIU6, SB491U4, 168SU-8, and 168MIU9. All of these mutants either belong to the pyrX group or have a defect in OMP-DCase (pyrF). Mutants with other enzymatic defects did not accumulate orotate. Other pyrX mutants (SB270, SB305, and 168SU-3) died slowly on plates of tryptose blood agar, and orotate was not detected. S. typhimurium pyrE and pyrF strains, with deletions of OMP-PPase and OMP-DCase, respectively, also display this accumulation of orotate. Attempts to demonstrate a multi-enzyme complex. (i) Centrifugation experiments. When strain BK1 extracts made with lysozyme and BRIJ-35 were centrifuged at 100,000 x g for 4 h, increased specific activity in the sedimented material was observed for ATCase, DHOase, DHO-DHase, and OMP-PPase. When glycerol-stabilized extracts were used, a comparable increase in specific activity was noted for

these four enzymes accompanied by a similar increase in CPSase relative activity. (ii) Sucrose density gradients. Glycerolstabilized extracts of strain BK1 and pooled 2.0 M + 2.5 M ammonium sulfate cuts were used in sucrose density gradient experiments. CPSase, ATCase, and DHOase precipitated between 1.5 and 2.5 M (37 to 61% saturation); DHO-DHase precipitated between 2.0 and 3.0 M (49 to 73% saturation); most OMP-PPase and OMPDCase activity was lost during the ammonium sulfate treatment. With the resuspended 2.0 + 2.5 M ammonium sulfate precipitates. CPSase, ATCase, and DHOase activities were found in the same area of the gradient, and the regions of maximal activity all appeared to coincide (Fig. 2). The approximate molecular weight can be estimated at 130,000. Regulation studies. Further attempts to characterize the pyrimidine biosynthetic enzymes of B. subtilis were made by determining their response to a variety of related compounds previously reported to be involved in the regulation of the pyrimidine enzymes of other organisms (see reference 32 for review). The major point of regulation in B. subtilis appears to be CPSase, and the results of these studies on that enzyme are presented elsewhere (34). Unlike most other bacteria studied to date, no inhibition or activation could be determined in in vitro assays of ATCase activity in B. subtilis. (i) Feedback inhibition and activation. Various compounds were tested for possible effects on each of the enzymes of the pathway leading to uridine 5'-monophosphate (UMP). The results are listed in Table 5, and the regulation of

TABLE 3. Enzyme activities of the mutantsa Strain

|

H-37

168MIU5 168MIU8 168MIU16 168SU1

168MIU3 168MIU4 168MIU7 168MIU13 168MIU14 168MIU15 168MIU17 SB491U3 G-18 168SU2 168SU5 168MIU2 168MIU10 168MIU12 168MIU18 SB491U1 SB491U6 SB491U7 SB491U8 GSY289 MB102 SB319 SB8 168MIU9 168SU7 WB577 17A-42 168MIU1 SB491U2 BR72 27-6

168MIU11 168MIU6 SB491U4 168THY-U168SU3 SB270 SB5d BD71d MB45d MB46d MB106d MB169d SB305 AU UTT A26U 168TUT SB491U5 S. typhimurium pyrA81

CPSase

ATCase

0.Olb 15.70 3.40 2.66 0.55 8.09 2.03 3.36 10.87 6.71 6.59 1.42 1.27 1.60 1.70 0.09 7.52 12.67 14.11 18.80 3.37 55.95 22.92 0.92 4.32 1.15 1.09 3.13 8.47 1.00 0.20 0.73 2.71 0.30 1.42 2.71 5.00 0.31 50.52 4.04 2.06 3.42 2.39 0.41 0.71 2.42 1.62 0.45 0.71

78.2 0.00 0.00 0.85 0.00 246.4 255.4 178.8 143.7 158.8 140.0 202.2 230.6 163.3 144.0 93.5 260.5 149.5 133.2 117.0 189.6 117.1 88.5 238.4 78.2 90.12 134.4 82.5 190.8 227.0 0.26 133.4 202.2 93.8 108.7c

132.Oc 0.00 184.4

0.00 0.00

138.5 97.7 89.8 118.6 237.5 70.0 258.9 190.0 149.5 175.3 125.6 224.0 171.8 112.9 0.00 91.4

0.00

NT'

1.21 2.70e

4.64e

Enzyme activity 1_DHOase j DHO-DHase J OMP-PPase

OMP-DCase

9.39 14.70 15.00 16.20 6.71 0.13 0.63 0.00 0.00 0.74 0.00 0.00 0.61 0.09 0.00 0.00 6.81 11.20 10.80 9.23 16.17 9.93 5.06 11.63 28.99 8.12 6.02 6.12 6.18 14.06 7.44 0.00 4.46 6.11 6.13c 7.26c 1.45 8.17 20.00 6.35 7.96 8.76 13.87 4.44 20.23 16.94 11.72 3.36 11.40 11.10 11.33 4.77 1.70 0.00

3.74 7.65 9.21 6.73 0.00 8.31 6.32 4.45 6.62 4.33 5.76 3.99 5.64 8.24 5.30 3.14 0.00 0.00 0.00 0.00 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.40 5.17 12.00c 0.00 0.00 1.05 0.00 4.17c 0.00 3.92 4.12 1.02 0.80 3.94 5.82 1.94 9.89 8.07 5.11 4.69 5.39 1.37 0.88 1.58 2.58 0.63

0.89 2.07 1.75 1.21 0.70 0.91 1.84 1.66 1.09 1.02 0.90 1.40 1.54 1.03 0.79 0.81 1.73 1.87 1.28 1.41 0.94 0.66 0.92 1.42 1.17 0.40 1.26 2.78 1.38 1.33 0.71 1.67 1.32 3.52 1.26 0.92 2.20 1.26 0.23 1.70 0.70 0.75 1.10 1.02 0.32 0.22 0.53 0.52

0.89 1.03 1.06

171.72 13.59

9.38

NT

NT

NT

1.17 1.00 1.45

49.88 35.44 86.71 57.16 26.01

60.87 94.01 83.95 70.72 58.24 39.43 43.32 64.30 77.93 77.39 67.55 41.84 61.94 71.90 214.71 49.98 40.06 34.20 65.36 41.85 40.49 12.08 3.70 0.00 0.00 29.17 14.62 0.00 0.00 57.85 2.97 17.53 73.96 13.40 61.90 22.18 39.98 49.77 24.93 80.89 94.67 32.05 25.08 69.96 29.16 33.82 18.62

0.72 0.26 NT NT NT NT pyrB137 NT 373.8 1.79 NT NT NT pyrC66 NT NT NT 0.00 NT NT pyrD67 NT NT NT 0.00 NT 5.99 pyrE125 NT NT j NT 0.26 7.68 ptrF146 0.00 a Enzymatic activities are expressed as described in Table 2. "These assays were done on cells grown under arginine- and uracil-derepressed conditions. cThese activities are unstable and have previously been found to be zero. The detection of activity apparently depends on slight variations in growth, extraction, or assay techniques. d

These strains all have the same ura- mutation.

Activity for these strains could be demonstrated only in the presence of activator (10 gmol of PRPP). ' NT, Not tested. e

609

610

J. BACTERIOL.

POTVIN, KELLEHER, AND GOODER TABLE 4. Classification of pyrimidine-requiring mutants

Class

Mutantsa

CPSase

pyrA pyrB

H-37 M-5; M-8; M-16; SU-1

pyrC

G-18; M-3; M-4; M-7; M-13; M-14; M-15; M-17; S-3; SU-2; SU-5 SB319; SB8; GSY289; GSY318; M-2; M-10; M-12; M-18; S-1; S-6; S-7; S-8; MB102 M-9; SU-7 WB577

pyrD

pyrF pyrDHb

ATCase

DHOase DHO-DHase OMP-PPase

OMP-DCase

Mutants in class

+

+

+

1

+

+

+

4

+

+

+

+

11

+

+

_

+ +

+

+

+

_

+ _ +

-(+) _ _

+ + +

+ +

-

-

+

_

+

+

13

17A-42 M-1; S-2

+

pyrDF

+

+ _ + +

PYrDBCC

27-6; BR-72;

+

-(±)

-(±)

-(+)

+

+

2 1 1 2 3

pyrX

SB270; SB5;

+

+

+

+

+

+

11

_

_

+

±

+ +

1 1

pyrDc

M-11

_

SB305; AU; UTT; A26U;

M-6; S-4; 168THY -U-; SU-3; SU-8 pyrABC pyrACD

168TUT S-5

_

+

+

a Strains designated "M-" are from the 168MIU series; strains designated "S-" are from the SB491U series; and strains designated "SU-" are from the 168SU series. bDHO-DHase activity unstable-depends on slight variations in growth and extraction. c ATCase, DHOase, DHO-DHase activities unstable-depends on slight variations in growth and extraction.

the pathway as a whole (including CPSase) is schematically presented in Fig. 3. Especially noteworthy are the activation of CPSase by ornithine and PRPP; the inhibition of the same enzyme by UTP, dihydroorotate and avidin; the inhibition of DHO-DHase by orotate, carbamyl aspartate, guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP); and the inhibition of OMP-PPase by UMP, UTP, adenosine 5'-triphosphate (ATP), and dihydroorotic

I>

04

0. v)

02

D0 0

FRACTION NO.

FIG. 2. Cosedimentation of three pyrimidine biosynthetic enzymes in 5 to 20% sucrose density gradients. Abbreviations used: CATase, catalase (beef liver, molecular weight 247,000); CPSase, carbamyl phosphate synthetase; ATCase, aspartate transcarbamylase; DHOase, dihydroorotase. The specific radioactivity of the diluted bicarbonate solution for the CPSase assay was 205 counts/min per nmol.

acid. (ii) Induction. It was not possible, under our growth conditions, to force wild-type B. subtilis to grow on any of the pathway intermediates. However, a few of the mutants would grow on orotic acid, and extracts from these strains could be used to ascertain whether any induction of the pyrimidine pathway enzymes had occurred. Strain BR72, which is defective in DHO-DHase, was grown with orotic acid and under uracil-derepressed conditions. Lysozyme extracts were prepared and levels of four enzymes were compared. Significant increases in

PYRIMIDINE BIOSYNTHETIC PATHWAY OF B. SUBTILIS

VOL. 123, 1975

611

the specific activities of DHOase and OMPCompetent recipient cells were transformed PPase were observed (1.7-fold and 4.2-fold, separately, with deoxyribonucleic acid from respectively); however, we were unable to ob- wild-type cells and from each mutant strain. tain convincing evidence for induction. The calculated recombination indices from (iii) Repression. Strain BK1 shows a drop in transformation experiments with strains SB5 activity for all six enzymes of the pathway when and SB319 are presented in Table 6. The grown in 5 gg of uracil per ml (Table 2). linkage of the CPSasepyr mutation (pyrA) in DHOase, DHO-DHase, and OMP-DCase activ- strain H-37 (Table 1) to the mutations of strains ities fall to lower levels when the cells are grown SB5 and SB319 with co-transformation techin medium containing a 10-fold-higher concen- niques was previouslv reported (34). Only a tration of uracil. general ordering of groups of similar biochemiGenetic characteristics of the mutants. In cal mutations, and not an assignment of precise order to expand the linkage map of B. subtilis, map positions, is intended. the same two strains used in the previous Correlation of biochemical and genetic studies, SB5 and SB319, were used as the data. The genetic map derived from the transrecipients in transformation experiments (23). formation experiments is represented together TABLE 5. Inhibition and activation of the pyrimidine biosynthetic enzymes from strain BKI Relative activities Effectora

Concn (umol)

ICPSase None UMP UMP UTP UTP UTP Arg and UTP CTP Dihydroorotate Dihydroorotate Carbamyl phosphate Carbamyl phosphate Carbamyl phosphate ATP ATP IMP IMP AMP AMP GMP Orotate Orotate Orotate OMP OMP Carbamyl aspartate PRPP Ornithine Arg Arg ACGLN Uridine Biotin Avidin

IATCase

I

1.00 5 10 5 10 20 10 each 5 10 100 5 10 100 2 10 2 10 5 10 10 1 2.5 5 0.55 5 10 10 10 10 100 10 100 10 1 unitc

DHOase DHO-DHaseb OMP-PPaseb OMP-DCase

I~~~~~~~10

1.00 1.04

1.00 0.80

1.02

0.48

0.58 0.18

1.00

1.00

0.81

0.10

0.62

0.20

1.00

0.31 0.01

0.69 0.18

1.07 0.84

0.76

0.47

0.83

0.84 0.94 0.45

0.95 0.91

0.39

0.51

0.38

0.95

0.57

0.02

0.81

0.96 0.94

0.60 0.72

1.58 0.00 0.54

0.62

1.14

1.23 0.90 1.41

0.86 1.05

0.86

1.05

0.33

0.70 0.45

1.00 0.87

0.89

3.76 2.05 1.09 0.87 1.54 0.72 0.84 0.10

aAbbreviations used: ACGLN, acetyl-glutamate; Arg, arginine; AMP, adenosine-5'-monophosphate, ATP, adenosine-5'-triphosphate; CTP, cytidine-5'-triphosphate; GMP, guanosine-5'-monophosphate; IMP, inosine-5'-monophosphate; OMP, orotidine-5'-monophosphate; PRPP, 5-'phosphoribosylpyrophosphate; UMP, uridine-5'-monophosphate; and UTP, uridine-5'-triphosphate. BK1 lysozyme extract not centrifuged. c 1 unit of avidin will bind 1 ,g of D-biotin.

612

POTVIN, KELLEHER, AND GOODER

J. BACTERIOL.

1) provide additional support for this assignment. The map is merely meant to suggest the likely order of the groups of mutations and should not be construed as giving a precise map position for each individual mutation. However, it does appear that all the known pyrimidine mutations of B. subtilis fall in an area of the genome no larger than a single transformation unit. This would correspond to a piece of deoxyribonucleic acid having a molecular TABLE 6. Transformation data Donor

CITRULLINE

I-AR61tJlt4E L-ARGININE . UTP_ SUCCINATE ---.UDP

- ARGININE

_:_

Recombination indexa Recipient Recipient (SB5) (SB319)

- -_

CTP

FIG. 3. Regulation of the pyrimidine biosynthetic pathway in B. subtilis. Key: (-----) activation; (----) repression; and ( ) inhibition.

with the enzymatic deficiencies of the mutants in Fig. 4. It can be seen that most mutants with similar enzymatic defects are closely linked on the map. Unlike other organisms studied previously, in B. subtilis the loci for all pyrimidine biosynthetic enzymes appear to be contiguous. With the exception of two strains (SB491U5 and 168TUT), all mutants with multiple enzymatic defects lie within the pyrD (DHO-DHase) locus.

BK1 168MIU1 168MIU2 168MIU3 168MIU4 168MIU5 168MIU6 168MIU7 168MIU8 168MIU9 168MIU10 168MIU11 168MIU12 168MIU13 168MIU14 168MIU15 168MIU16 168MIU17 168MIU18 SB491U1 SB491U2 SB491U3 SB491U4 SB491U5 SB491U6 SB491U7 SB491U8 168SU- 1 168SU-2 168SU-3 168SU-5 168SU-7 168SU-8 GSY289 GSY318 G18 168THY-U MB45 MB46 MB102 MB106 MB169 BD71

1.000

0.108 0.150 0.567 0.343 0.516 0.104 0.471 0.321 0.147 0.156 0.146 0.166 0.405

0.374 0.392 0.497 0.398 0.192 0.218 0.132 0.402 0.094 0.398 0.146 0.252 0.281 0.486 0.363 0.033 0.405 0.121 0.042

1.000 0.034 0.000 0.407

0.283 0.211 0.101

0.336 0.202 0.076 0.032 0.040 0.021 0.391 0.402 0.320 0.235 0.315 0.029 0.000 0.053 0.473 0.227 0.493 0.112 0.110 0.091 0.251

DISCUSSION All of the pyrimidine auxotrophs (Table 1) used in this study were derived from B. subtilis strain "168MI." The lineage and methods of mutagenesis are clear for all the newly isolated strains, but unfortunately this information is difficult or impossible to obtain for some of the original mutants obtained from other laboratories. The results of the transformation experi0.396 ments with the two recipients, strains SB5 and 0.161 SB319, varied. The genetic map (Fig. 4) was 0.295 generally constructed using transformation 0.132 data from strain SB5 for those markers falling 0.234 in the right third of the map, and data from 0.190 0.129 strain SB319 crosses for those in the left two0.194 0.014 thirds. It was assumed that the more closely the 0.564 0.260 recipient and donor markers were physically 0.003 0.308 associated, the more accurate the recombina0.151 tion index data were likely to be. The pyrA locus 0.149 0.175 0.007 is thought to be located at the extreme left side 0.000 of this map on the basis of the positions of the 0.000 0.189 multiple mutations pyrABC (strain 168TUT) 0.000 0.153 and pyrACD (strain SB491U5). Previously reported co-transformation data (34) concerning aRecombination index was calculated according to the CPSasepyr mutation of strain H-37 (Table the method of Kelleher and Gooder (23).

PYRIMIDINE BIOSYNTHETIC PATHWAY OF B. SUBTILIS

VOL. 123, 1975

613

M-6 MI SsM

SU-7

M102 M-4 GI18 15 M-15Ms-1I7

S-5

168TUT S-3

PYR ABC PYR ACD

M-IM4 M3

S-16M-5

~~~~WB577 -

-

5Y8 MM

PYR C

M-9

SB305

M-2 8319

S-2

UTT

PYR 8

'-PYR A

PYR D PYR DB PYR DC PYR DS PYR DX

AU168THY-u -

{ I

}

S-S S8270

I

76

BR-72

SB5

M-I

MBS S-7S fj{jSjjj{jMj1f I~ ~ ~

17A42

SU-2

A

SI

-1

A26U

PYR

PYR

X

PYR

DF

FIG. 4. Correlation of the genetic data with the enzymatic deficiencies of the pyrimidine-requiring mutants. Strains designated "M-" are from the 168MIU series; strains designated "S-" are from the SB491 U series; and strains designated "SU-" are from the 168SU series. The letters following "pyr" correspond to deficient enzymes in the pyrimidine pathway creating a pyr requirement as follows: A, CPSase; B, A TCase; C, DHOase; D, DHO-DHase; F, OMP-DCase; and X, unknown.

weight of approximately 1.5 x 107 or 0.38% of the entire genome (2, 11). In the cases of the two possibly rate-limiting steps of the pathway, CPSase and OMP-PPase, the enzymatic activities were reproducible although usually quite low. The strains that have been found to lack detectable pyrimidinespecific CPSase activity (SB491U5, H-37, and 168TUT) have been retested at least 10 times. The S. typhimurium CPSase deletion mutant (pyrA81) has been observed to yield quantitative results that are very similar to those observed for the B. subtilis mutants mentioned above. It is not possible to determine the sensitivity of this assay procedure, unless partially purified carbamyl phosphate synthetase becomes available. Previous work in this laboratory has demonstrated that the OMP-PPase activity from B. subtilis deviated from linearity within one to three minutes. The possibility that this problem might be a result of feedback inhibition by OMP, the product of the enzyme (reported for E. coli B by Lieberman and Simms [25]), is not substantiated by our observation that the addition of purified yeast OMP-DCase does not prolong the B. subtilis OMP-PPase linear activity. An attractive possibility would be that some factor, either a membrane fragment or some high-molecular-weight component normally associated with the membrane, is undergoing a separation from the enzyme involving centrifugation-induced changes in enzyme conformation such as might be inferred from the increased period of linearity observed when crude extracts were centrifuged for 60 min instead of 30 min (see Results). During her work with the membrane-bound E. coli DHO-DHase, Karibian (20) discovered that Triton X-100 could restore DHO-DHase activity that had been destroyed by treatment with phospholipase A2. In E. coli, BRIJ-35,

Triton X-100, and bovine serum albumin can all substitute to some extent for lost lipids to restore enzymatic activity (20, 21). Our observations concerning the sedimentation behavior of some of the pyrimidine biosynthetic enzymes in B. subtilis may be viewed as suggestive evidence of a particulate association of these enzymes. Further evidence for possible membrane attachment was obtained from studies on CPSase (34). The results of the sucrose density gradient demonstrated that CPSase, ATCase, and DHOase exhibited maximal activity at precisely the same location in the gradient (Fig. 2). Similar results had previously been obtained in mammalian cells (38) and in Serratia marcescens (Wild, Ph.D. dissertation, University of California, Riverside, 1972). In both of these cases, an enzyme complex is thought to exist. In mammalian cells, the CPSase-ATCase-DHOase complex has a molecular weight of approximately 750,000 to 850,000, whereas the last two enzymes, OMP-PPase and OMP-DCase, form a second aggregate with a molecular weight of 100,000 to 140,000 (Fed. Proc. 31:473, 1972). The results reported here for the first three pyrimidine enzymes of B. subtilis indicate a molecular weight of approximately 130,000. Of the twelve classes of enzymatic defects observed in the pyrimidine mutants of B. subtilis (Table 4), only five have been previously described in other organisms. Six of the others, composed of eight mutants, display multiple enzymatic defects under our in vitro assay conditions. The genetic evidence shows that six of these strains contain mutations which are located within the pyrD (DHODHase) locus (Fig. 4), and the biochemical data indicating unstable activity for some of the defective enzymes (Table 3) led to speculation concerning the nature of these mutations. An examination of the various combinations of

614

POTVIN, KELLEHER, AND GOODER

enzymatic defects rules out classical polarity eff'ects. The observed reversion frequencies of 10- to 10-9 for these same six strains provide negative evidence for deletions or multiple mutations. Reversion of the other two mutants, strains 168TUT and SB491U5 (Table 1), which lie some distance from the pyrD locus, has not been observed. The most likely explanations for these multiple defects would seem to be that either a single mutation is altering the conformation and activities of' more than one enzyme, or a defect in a single enzyme produces a pleiotropic effect on other enzymes through some complex regulatory mechanism. Note, however, that mutants with a defective DHODHase do not always have other enzymatic deficiencies. One of the most interesting classes of mutants, pyrX, includes seven strains. All attempts to demonstrate an enzymatic defect with in vitro methods have been unsuccessful. The massive excretion of orotate by most pyrX mutants, along with the other observations mentioned in the Results section, leads one to conclude that pyrX mutants may lack in vivo (and possible in vitro) activity for either OMPPPase or OMP-DCase. On the basis of the locations of their mutations (directly adjacent to the OMP-DCase locus) and the absence of any known B. subtilis OMP-PPase mutants, it is possible that they may be leaky pyrE mutants. Additional support for this theory was recently obtained when three of' the pyrX mutants (strains A26U, SB5, and SU-8) were retested for OMP-PPase activity by using a slight modification of the method of Shoaf and Jones (38) and were found to have less than 4% of the activity of wild-type strain BK1. The possibility that pyrX mutants may have defective pyrimidine-specific PRPP synthetase activity can also not be ruled out. Unlike the enzyme of most enteric bacteria, the B. subtilis ATCase is insensitive to feedback control. The CPSase is, however, subject to both feedback inhibition and activation (Fig. 3). The regulatory consequences of these properties as well as the interaction of CPSase with proteins such as avidin and lysozyme have been previously discussed (34). It is interesting to note that UTP has a moderately inhibitory effect on DHOase in addition to its much stronger action on CPSase. This could arise due to the multi-enzyme complex suggested by the sucrose density gradient work. Along the same line of reasoning, CPSase is somewhat (-5O0%) inhibited by the product of DHOase. The other major sites of inhibition in the pyrimidine

J. BACTERIOL.

pathway are DHO-DHase (by orotate) and OMP-PPase (by UMP). One f'inal point is pertinent. In B. subtilis, the balanced (steady-state) production of purines and pyrimidines is partially accomplished by PRPP concentration. PRPP is used as a substrate by both the purine and pyrimidine pathways, is a powerf'ul activator of CPSase (34), and inhibits adenosine-5'-monophosphate synthetase (17). In S. typhimurium, the enzyme that synthesizes PRPP. phosphoribosylpyrophosphate synthetase, is repressed by uridine compounds (33). Additional control of the enzyme is provided in B. subtilis by IMP and GMP, which strongly inhibit DHO-DHase (Fig. 3; Table 5). On the basis of the information gathered during this study, a multienzyme complex in which DHO-DHase is the central enzyme is suspected to exist in B. subtilis. The five other enzymes are most likely associated with the central enzyme in two groups: CPSase-ATCaseDHOase and OMP-PPase-OMP-DCase. A similar complex was previously proposed for B. subtilis by Kelleher (Ph.D. dissertation, University of North Carolina, Chapel Hill, 1969) and for mammalian cells by Shoaf and Jones (M. E. Jones, personal communication). This model seems to fit most of the available data, and makes it possible to visualize how a mutational alteration in the configuration of the DHO-DHase enzyme could cause a loss of activity in anv of the five other enzymes. ACKNOWLEDGMENTS We wish to thank Kenneth Bott and Robert Twarog who generously provided the use of their equipment, expertise, and advice, and Gerard O'Donovan for his many helpful suggestions and his critical reading of this manuscript. This investigation was supported by a United Medical Foundation grant 324UNI (256) and also by grant AI-04577 from the National Institute of Allergy and Infectious Diseases. B. W. P. and R. J. K. were supported by National Institute of General Medical Services grant GM006-85. LITERATURE CITED 1. Appel, S. H. 1968. Purification and kinetic properties of brain orotidine-5'-phosphate decarboxylase. J. Biol. Chem. 243:3924-3929. 2. Armstrong, R. L., N. Harford, R. H. Kennett, M. L. St. Pierre, and N. Sueoka. 1970. Experimental methods for Bacillus subtilis, p. 36-59. In H. Tabor and C. W. Tabor (ed.), Methods in enzymology. Academic Press Inc., New York. 3. Barratt, R. W., and A. Radford. 1970. Linkage maps of Neurospora crassa, p. I 68-I 78. In H. A. Sober (ed.), Handbook of biochemistry with selected data for molecular biology, 2nd ed. Chemical Rubber Co., Cleveland. 4. Beckwith, J. B., A. B. Pardee, R. Austrian, and F. Jacob.

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