pyrimidine biosynthetic pathway bacillus subtilis · 2006. 3. 10. · pyrimidine biosynthetic...

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

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

Pyrimidine Biosynthetic Pathway of Bacillus subtilisBARRY W. POTVIN,1* RAYMOND J. KELLEHER, JR.,2 AND HARRY GOODER

Curriculum in Genetics and Department of Bacteriology and Immunology, School of Medicine, University ofNorth 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 ofBacillus subtilis. The observed enzymatic deficiencies allowed the mutants to beplaced into 12 classes, some of which represent defects in more than one of the sixknown pyrimidine biosynthetic enzymes. Mapping analysis by transformationhas 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 hasbeen made. Those mutations conferring similar enzymatic deficiencies werefound to be contiguous on the B. subtilis map. Regulatory aspects of thepyrimidine pathway have also been investigated and are compared to previouslyreported results from other organisms. Evidence is presented which bears uponthe possible physical association of the first three enzymes and the association ofat 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 dihydroorotatedehydrogenase as the central enzyme in a proposed aggregate.

In previous publications (23, 34) we havepresented genetic evidence for a tightly linkedgroup of mutations affecting the pyrimidinebiosynthetic pathway of' Bacillus subtilis andhave described some biochemical and geneticaspects of the first enzyme of' that pathway,carbamyl phosphate synthetase (CPSase, EC2.7.2.5).These studies of' the pyrimidine biosynthetic

pathway (Fig. 1) in B. subtilis, a gram-positiverod, were undertaken to test comparative regu-lation between organisms. B. subtilis offers theadvantages of' its ability to grow on a well-defined minimal medium and the availability of'previously established methods for genetic anal-ysis (2). The biochemical properties of' B.subtilis have revealed that it is not only quitedifferent from Escherichia coli but also frommany other bacteria. A major site of' regulationin the pyrimidine pathway of many bacteriaseems to be aspartate transcarbamylase (ATC-ase, while in some Pseudomonas and Sac-charomyces uridine-5'-triphosphate (UTP) isphate (CTP) is the feedback inhibitor of ATC-ase; while in some Pseudomonas and Sac-charomyces, uridine-5'-triphosphate (UTP) isthe negative effector. Nucleotides do not haveany significant effect on the ATCase fromNeurospora or mammalian cells (7, 8, 14). The

'Present address: Department of Human Genetics andDevelopment, Columbia College of Physicians and Surgeons,630 W. 168th St., New York, N.Y. 10032.

2Present address: Salk Institute, P.O. Box 1809, SanDiego, Calif. 92112.

ATCase of B. subtilis is similar to the enzymefound in this latter group, being unaffected byvarious pyrimidine and purine nucleotides up toconcentrations of 1 mM (6, 35). It has also beenfound that the B. subtilis ATCase undergoes arapid, energy-dependent inactivation in sta-tionary-phase cells just prior to sporulation (9,40).

Existing data on the regulation of the activi-ties of the pyrimidine biosynthetic enzymes inB. subtilis afforded the conclusion that thecontrol of the pathway is the same as in othermicroorganisms; however, dihydroorotase(DHOase, EC 3.5.2.3) and carbamyl phosphatesynthetase (CPSase, EC 2.7.2.5) were not s.tud-ied (L. J. Rebello and G. A. O'Donovan, Tex.J. Sci. 24:117, 1972). In the pyrimidine pathwaysof Saccharomyces and Neurospora a complexcomposed of' CPSase and ATCase has beenobserved (18). Mammalian cells have beenreported to possess complexes of CPSase-ATCase-DHOase and orotidine-5'-monophos-phate pyrophosphorylase (OMP-PPase, EC2.4.2.10)-orotidine-5'-monophosphate decar-boxylase (OMP-DCase, EC 4.1.1.23) (1, 14, 19,22, 37, 38; Fed. Proc. 31:473, 1972). In Serratiamarcescens, 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 ofCalifornia, Riverside, 1972). Evidence for theexistence of a similar multi-enzyme complex inB. subtilis as originally suggested by R. J.Kelleher (Ph.D. dissertation, University of

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PYRIMIDINE BIOSYNTHETIC PATHWAY OF B. SUBTILIS

HOOC

CH2

.olCH MO 0H2N N"COOH N.,\ /°M2 ATCose C~

L-Asportic acid NM72 H2 DHOos HN lCH2 DHO-DHCM PYRC -0CIN. ~CH PYR

00O00 "N-'N.oCOOH 0 N "CHC00H-1. H2N-COPO3H2 Corbomyl-L- L-Dihydroorotkc ocid

RA carbomyl phosphate Asportic ocid

?I ° OIMP-INJCLEIC UTP MHN' NCH HN CACIDS-CTP

OT N PYR F 0 C CCOOHribo4-5'-P rib-5'-P

Undlne-5'-Phosphate Orotidne-5- Phosphate

011sC-

MN NC

11HH

PRPP Orotic acid

PYR E

FIG. 1. The de novo pyrimidine biosynthetic pathway.

North Carolina, Chapel Hill, 1969) is presentedin this paper.

Genetic data on pyrimidine-requiring mu-tants of B. subtilis obtained in this and previousstudies indicated that all known Pyr- muta-tions were linked (10, 23, 42). This is unique andunlike the situation for any other microorga-nisms on which genetic data have been pub-lished (E. coli [39]; S. typhimurium [36];Klebsiella pneumoniae [28]; Pseudomonasaeruginosa [16]; Saccharomyces cerevisiae [24];Streptomyces coelicolor [15 ]; Diplococcus pneu-moniae [29]; Coprinus radiatus [12]; Neuro-spora crassa [3 ]).

MATERIALS AND METHODSBacterial strains. Sources of the strains of B.

subtilis, S. typhimurium, and E. coli used in thisstudy are listed in Table 1.

Mutagenesis and selection procedures. Ni-trosoguanidine mutagenesis and penicillin selectiontechniques were as previously described (34). Al-though the selection technique was also designed toisolate mutants with a dual requirement for botharginine and uracil ("AU"), none was found amongthe approximately 14,000 colonies checked. The Pyr-mutants that were isolated are most probably theresult of single-point mutations because of the ob-served spontaneous reversion frequencies which wereusually in the range of 10-7 to 10-g.

Chemicals, media, and growth conditions. Thesources of chemicals, preparation of media, andgrowth conditions were described in previous publica-tions (23, 34). All mutant strains were grown underderepressed conditions (the incubation of cultures for2 h after growth ceased in a defined minimal mediumwith a limiting concentration of 5 lAg of uracil per ml[34]). Strain BKl was also grown under arginine-repressed (50 ug of arginine per ml) or uracil-repressed(50 IAg of uracil per ml) conditions and in the presenceof 5 gg of uracil per ml. The other wild-type strains-12A, 168MI-, W23, and SB491-were grown in theabsence of added uracil or arginine.Growth on intermediates. Attempts were made to

obtain growth of uracil-requiring mutants on three ofthe intermediates in the pathway. Liquid minimalmedium supplemented with 300 Mg of L-dihydrooro-tate per ml, with 350 Mg of carbamyl aspartate per ml,with 120 sg of orotic acid per ml, or with 50 Mg ofuracil per ml was used for this purpose. Unsupple-mented minimal medium was used as a control.Growth was monitored by using a calibrated Klettcolorimeter with a no. 54 filter.

Experiments were also conducted on solid minimalmedium supplemented as above.

Preparation of extracts. Methods used to prepareextracts have been described previously (34). Glyc-erol-stabilized extracts contained 10% (wt/vol) glyc-erol and 1 mM dithiothreitol.

Cell extracts prepared by lysozyme treatment withthe addition of BRIJ-35 (final concentration = 1%)were assayed for enzyme activities with the exceptionof CPSase activity, which was usually determinedwith "Damaged Cell Preparations" (34). In somecases noncentrifuged preparations were used to con-firm 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 fromstrains of S. typhimurium reputed to contain individ-ual deletions for each of the six enzymes of thepyrimidine biosynthetic pathway (41; O'Donovan,personal communication). Extracts of E. coli B wereused to provide an additional control for each assay.All enzyme assays were performed at 30 C with theexception of CPSase, which was incubated at 37 C.

(ii) CPSase. This enzyme was assayed by themethod of Potvin and Gooder (34).

(iii) ATCase. This assay was made according tothe procedure of Neumann and Jones (30) with minormodifications. Dilithium carbamyl phosphate wasobtained from Sigma Chemical Co. and required nofurther purification. The amount of product formed inthis reaction was determined with the method ofPrescott and Jones (35).

(iv) DHOase. This enzyme was assayed in thereverse direction by using a slight modification of themethod of Beckwith et al. (4). The amount of productwas determined in the same way as for ATCase.

VOL. 123, 1975

ATP CPSGLUTAMINE

HCOj PYI

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606 POTVIN, KELLEHER, AND GOODER

TABLE 1. Bacillus subtilis stock culturesa

Strain Source Parent Phenotype nutritionaldesignation jSuc strain j requirements168MI168T+SB491W23ASP12ABK1168MIU1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18SB491U1SB491U2, 3, 4, 5,c 6, 7, 8168SU-1, 2, 3, 5c, 7c, 8cH-37H-59SB270SB5BD71MB45MB46MB102MB106MB169SB305AUUTTA26UcSB319cSB8cBR7227-6WB57717A-42168TUTC168THY -UG18GSY289GSY318Other bacterial stocks

E. coli BS. typhimuriumpyrA81pyrB137pyrC66pyrD67pyrE125pyrF146

SpizizenBott (W23 x 168MW'CarltonSpizizenSpizizenTL(168T+ x 168MW)5TL (NTG)

TL (NTG)TL (NTG)Spizizen (EMS)Jensen (NTG)Jensen (NTG)NesterNesterDubnauMarmurMarmurMarmurMarmurMarmurNesterYoungYoungMarmurNesterNesterYoungGreer (OHNH2)Nester (UV)Greer (HNO2)Romig (UV)Bresler (Thy starv)GreenAnagnostopoulosAnagnostopoulos

Meynell

O'DonovanO'DonovanO'DonovanO'DonovanO'DonovanO'Donovan

168MI168MI

168MI168MI168MI

SB491SB491168MIWB746A139168MI168MISB3168MI168MI168MI168MI168MI168MI168MI9

168MI168MI168MI168MI168MI168MI168MI168MI168MI168MI168MI168MI

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

TrpNoneNoneNoneNoneNoneTrp Ura

Trp UraUraTrp UraNone (Arg")None (Ural)UraTrp Ura HisTrp Ura HisTrp Ura HisTrp Ura His LeuThy Pur UraMet UraTrp Ade UraMet UraArg UraTrp Thr UraUraMet UraThr UraTrp UraUraUraUraTrp Thy UraThy UraTrp UraTrp UraTrp Ura

None

Arg UraUraUraUraUraUra

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 or168T+.

c These strains were never observed to revert.

(v) Dihydroorotate dehydrogenase (DHO-DH- that of Beckwith et al. (4). The reaction mixture inase, EC 1.3.3.1). The modification described by total volume of 1.2 ml contained 100 ,mol of tris(hy-O'Donovan and Gerhart (31) of the procedure of droxymethyl)aminomethane-hydrochloride, pH 8.5,Beckwith et al. (4) was adopted for this assay. 0.25 gmol of orotic acid, 2.5 ,umol of magnesium

(vi) OMP-PPase. This assay was modified from chloride, and 0.5 ,umol of tetra sodium 5'-phos-

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PYRIMIDINE BIOSYNTHETIC PATHWAY OF B. SUBTILIS 607

phoribosylpyrophosphate (PRPP, Calbiochem). Con-version of orotic acid to orotidine-5'-monophosphate(OMP) was monitored spectrophotometrically with aGilford 2000 recording spectrophotometer as a func-tion of the decrease in absorbance at 295 nm. To asilica cuvette (1-cm light path, 1.2-ml working vol-ume), buffer, orotic acid, magnesium chloride, anappropriate volume of distilled water, and 0.1 ml ofextract were added. The optical density at 295 nm wasmonitored to determine if any endogenous pyrophos-phorylase activity was present. The reaction wasinitiated by the addition of PRPP. The absorbancechange during the initial linear portion of the reactioncurve was determined. An optical density decrease of3.67 units corresponds to the conversion of 1 Mmol oforotic acid (4).

(vii) OMP-DCase. The technique used for thisassay was a modification of the method of Beckwith etal. (4). The reaction mixture contained 100 smol oftris(hydroxymethyl)aminomethane-hydrochloride, pH8.5, 0.275 Mimol of OMP, 2.5 gmol of magnesium chlo-ride, 0.1 ml of extract, and enough H2O to make atotal volume of 1.0 ml in a silica cuvette (workingcapacity 1.2 ml, path length 1.0 cm). The reaction wasinitiated by the addition of OMP. The resultingdecrease in absorbance at 285 nm was monitored on aGilson 2000 recording spectrophotometer which hadbeen set at zero absorbance by using water. A decreaseof 1.38 units corresponds to 1 Mmol of substratedecarboxylated.

(viii) Catalase. Beef liver catalase (molecularweight = 247,000, Worthington Biochemicals) wasused as a marker enzyme in sucrose density gradients.It was assayed by measuring the disappearance ofhydrogen peroxide spectrophotometrically at 240 nm(5).

(ix) Protein determinations. Protein concentra-tions of extracts and column fractions were deter-mined with a slight modification of the technique ofLowry et al. (26) with bovine serum albumin as thestandard.

Separation of enzymes. Ammonium sulfate frac-tionation, gel filtration, and sucrose density gradientcentrifugation were as previously described (34).

Transformation and construction of linkagemap. Deoxyribonucleic acid extraction, growth ofcompetent recipient cells, and transformation were aspreviously described (23, 34). The original linkagemap was confirmed and expanded by the samemethods used in earlier work (23).

RESULTS

Response to intermediates. Growth to nor-mal stationary-phase levels was obtained inliquid minimal medium supplemented withorotate using strains SB319, BR72, 17A-42,WB577, and 168TUT (Table 1). Dihydroorotateor carbamyl aspartate usually would not sup-port growth. In all cases no growth had occurredin unsupplemented minimal medium by thetime growth was recorded in the presence of theintermediate. All strains tested continued to

display their original uracil requirement aftergrowth on an intermediate.Preparation of extracts. It has previously

been reported in other microorganisms thatDHO-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 encounteredin assaying DHO-DHase in B. subtilis by usinglysozyme extracts that had been centrifuged at25,000 x g for 30 min. Centrifugation at 100.000x g for 4 h, however, destroyed DHO-DHaseactivity when lysozyme extracts had not beentreated with the nonionic detergent BRIJ-35. IfBRIJ-35 was used, the activity was 10-foldhigher in the 100,000 x g pellet than in thesupernatant fluid. It was also found that cen-trifugation at 25,000 x g for 60 min instead of 30min significantly prolonged the linear activityobserved in OMP-PPase assays. If the sedi-mented material was remixed with the superna-tant fluid, the reduced period of linearity wasobserved once again.Biochemical characterization of the

mutants. The levels of activity present inwild-type strains of B. subtilis are presented inTable 2, while similar data for the variousenzymes in the mutant strains are presented inTable 3. Enzymatic deficiencies are reproduci-bly observed and higher activities could not bedemonstrated when the amounts of substrate orextract were increased. Extracts from mutantswith enzymatic defects do not inhibit the corre-sponding activity in extracts of wild-type strainBK1. Several strains (BD71, MB45, MB46,MB106, MB169) have the same uracil mutationas strain SB5 (J. Marmur, personal communi-cation). These were included in this studybecause of a previous report that strain SB5 wasdeficient in ATCase activity (27). The mutantsare grouped in classes in Table 4 according totheir enzymatic defects.Attempts to determine the biochemical na-

ture of pyrX mutants. The identity of thebiochemical lesion in pyrX mutants proved tobe elusive. Earlier work in this laboratory haddemonstrated activity for the last five enzymesof the pyrimidine pathway (ATCase throughOMP-DCase) and implied that such mutantsmight lack a uracil-specific CPSase activity.The initial efforts to develop a CPSase assay(34) were aimed at testing this hypothesis. AllpyrX mutants had CPSase activity when grownunder arginine-repressed conditions (Table 3).In two strains, UTT and A26U (Table 3),CPSase was very low and difficult to detect. Inaddition, thiamine and citrulline were tested todetermine if the pyrimidine requirement of

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TABLE 2. Enzymatic activities of some wild-type strains

Enzyme activityStrain

CPSasea ATCaseb DHOaseb DHO-DHasee OMP-PPaseb OMP-DCaseb

168MI- 0.48 11.13 4.40 0.32 0.56 8.22SB491 0.73 17.86 3.04 0.52 0.71 9.45W23 0.72 17.88 4.98 1.79 1.19 6.34ASP12A 0.56 10.30 3.57 0.91 0.65 13.80BK1 1.00 23.30 2.01 0.41 1.49 16.51BK1J(U)d 0.00 0.00 0.54 0.13 0.42 3.40BK1-URAe 0.00 0.00 0.00 0.00 J 0.58 0.00

aRelative activity at 37 C, based on BK1, all strains arginine-repressed. One unit of relative activity isapproximately equal to 0.750 nmol of product formed/min per mg of protein. The specific radioactivity of thediluted bicarbonate was about 200 counts/min per nmol.

b The specific activities of ATCase, DHOase, OMP-PPase, and OMP-DCase are expressed as nanomoles ofproduct formed or substrate transformed per minute per milligram of protein at 30 C.

c One unit of DHO-DHase specific activity is that causing a change of 1.0 optical density unit at 480 nm permg 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.

these strains was a secondary effect of anenzymatic defect in another pathway, and itwas found that neither compound would sup-port the growth of pyrX mutants. Other possi-ble explanations include a deficiency in a pyr-imidine-specific phosphoribosylpyrophosphatesynthetase (PRPP synthetase, EC 2.7.6.1).Excretion of orotate. It was noted that after

prolonged storage of stock cultures on tryptoseblood agar plates (Difco) many of the pyrXmutants accumulated deposits of a white crys-talline material. This substance was identifiedas orotate on the basis of its absorption spec-trum and its ability to serve as the substrate forpurified yeast OMP-PPase (Sigma). This phe-nomenon was observed in strains SB5, UTT,A26U, AU, BD71, 168MIU6, SB491U4,168SU-8, and 168MIU9. All of these mutantseither belong to the pyrX group or have a defectin OMP-DCase (pyrF). Mutants with otherenzymatic defects did not accumulate orotate.Other pyrX mutants (SB270, SB305, and168SU-3) died slowly on plates of tryptose bloodagar, and orotate was not detected. S. typhimu-rium pyrE and pyrF strains, with deletions ofOMP-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 lysozymeand BRIJ-35 were centrifuged at 100,000 x g for4 h, increased specific activity in the sedi-mented material was observed for ATCase,DHOase, DHO-DHase, and OMP-PPase. Whenglycerol-stabilized extracts were used, a compa-rable increase in specific activity was noted for

these four enzymes accompanied by a similarincrease in CPSase relative activity.

(ii) Sucrose density gradients. Glycerol-stabilized extracts of strain BK1 and pooled 2.0M + 2.5 M ammonium sulfate cuts were used insucrose density gradient experiments. CPSase,ATCase, and DHOase precipitated between 1.5and 2.5 M (37 to 61% saturation); DHO-DHaseprecipitated between 2.0 and 3.0 M (49 to 73%saturation); most OMP-PPase and OMP-DCase activity was lost during the ammoniumsulfate treatment. With the resuspended 2.0 +2.5 M ammonium sulfate precipitates. CPSase,ATCase, and DHOase activities were found inthe same area of the gradient, and the regions ofmaximal activity all appeared to coincide (Fig.2). The approximate molecular weight can beestimated at 130,000.Regulation studies. Further attempts to

characterize the pyrimidine biosynthetic en-zymes of B. subtilis were made by determiningtheir response to a variety of related compoundspreviously reported to be involved in the regula-tion of the pyrimidine enzymes of other orga-nisms (see reference 32 for review). The majorpoint of regulation in B. subtilis appears to beCPSase, and the results of these studies on thatenzyme are presented elsewhere (34). Unlikemost other bacteria studied to date, no inhibi-tion or activation could be determined in invitro assays of ATCase activity in B. subtilis.

(i) Feedback inhibition and activation. Va-rious compounds were tested for possible effectson each of the enzymes of the pathway leadingto uridine 5'-monophosphate (UMP). The re-sults are listed in Table 5, and the regulation of

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TABLE 3. Enzyme activities of the mutantsa

Strain Enzyme activity| CPSase ATCase 1_DHOase j DHO-DHase J OMP-PPase OOMP-DCase

H-37 0.Olb 78.2 9.39 3.74 0.89 49.88168MIU5 15.70 0.00 14.70 7.65 2.07 35.44168MIU8 3.40 0.00 15.00 9.21 1.75 86.71168MIU16 2.66 0.85 16.20 6.73 1.21 57.16168SU1 0.55 0.00 6.71 0.00 0.70 26.01168MIU3 8.09 246.4 0.13 8.31 0.91 60.87168MIU4 2.03 255.4 0.63 6.32 1.84 94.01168MIU7 3.36 178.8 0.00 4.45 1.66 83.95168MIU13 10.87 143.7 0.00 6.62 1.09 70.72168MIU14 6.71 158.8 0.74 4.33 1.02 58.24168MIU15 6.59 140.0 0.00 5.76 0.90 39.43168MIU17 1.42 202.2 0.00 3.99 1.40 43.32SB491U3 1.27 230.6 0.61 5.64 1.54 64.30G-18 1.60 163.3 0.09 8.24 1.03 77.93168SU2 1.70 144.0 0.00 5.30 0.79 77.39168SU5 0.09 93.5 0.00 3.14 0.81 67.55168MIU2 7.52 260.5 6.81 0.00 1.73 41.84168MIU10 12.67 149.5 11.20 0.00 1.87 61.94168MIU12 14.11 133.2 10.80 0.00 1.28 71.90168MIU18 18.80 117.0 9.23 0.00 1.41 214.71SB491U1 3.37 189.6 16.17 0.05 0.94 49.98SB491U6 55.95 117.1 9.93 0.00 0.66 40.06SB491U7 22.92 88.5 5.06 0.00 0.92 34.20SB491U8 0.92 238.4 11.63 0.00 1.42 65.36GSY289 4.32 78.2 28.99 0.00 1.17 41.85MB102 1.15 90.12 8.12 0.00 0.40 40.49SB319 1.09 134.4 6.02 0.00 1.26 12.08SB8 3.13 82.5 6.12 0.00 2.78 3.70168MIU9 8.47 190.8 6.18 2.40 1.38 0.00168SU7 1.00 227.0 14.06 5.17 1.33 0.00WB577 0.20 0.26 7.44 12.00c 0.71 29.1717A-42 0.73 133.4 0.00 0.00 1.67 14.62168MIU1 2.71 202.2 4.46 0.00 1.32 0.00SB491U2 0.30 93.8 6.11 1.05 3.52 0.00BR72 1.42 108.7c 6.13c 0.00 1.26 57.8527-6 2.71 132.Oc 7.26c 4.17c 0.92 2.97168MIU11 5.00 0.00 1.45 0.00 2.20 17.53168MIU6 0.31 184.4 8.17 3.92 1.26 73.96SB491U4 50.52 138.5 20.00 4.12 0.23 13.40168THY-U- 4.04 97.7 6.35 1.02 1.70 61.90168SU3 2.06 89.8 7.96 0.80 0.70 22.18SB270 3.42 118.6 8.76 3.94 0.75 39.98SB5d 2.39 237.5 13.87 5.82 1.10 49.77BD71d 0.41 70.0 4.44 1.94 1.02 24.93MB45d 0.71 258.9 20.23 9.89 0.32 80.89MB46d 2.42 190.0 16.94 8.07 0.22 94.67MB106d 1.62 149.5 11.72 5.11 0.53 32.05MB169d 0.45 175.3 3.36 4.69 0.52 25.08SB305 0.71 125.6 11.40 5.39 1.17 69.96AU 1.21 224.0 11.10 1.37 1.00 29.16UTT 2.70e 171.8 11.33 0.88 1.45 33.82A26U 4.64e 112.9 4.77 1.58 0.89 18.62168TUT 0.00 0.00 1.70 2.58 1.03 171.72SB491U5 0.00 91.4 0.00 0.63 1.06 13.59S. typhimuriumpyrA81 0.00 NT' 9.38 NT NT NTpyrB137 0.72 0.26 NT NT NT NTpyrC66 NT 373.8 1.79 NT NT NTpyrD67 NT NT NT 0.00 NT NTpyrE125 NT NT NT NT 0.00 5.99ptrF146 NT NT j NT 0.26 7.68 0.00a 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.e Activity for these strains could be demonstrated only in the presence of activator (10 gmol of PRPP).' NT, Not tested.

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TABLE 4. Classification of pyrimidine-requiring mutants

Class Mutantsa CPSase ATCase DHOase DHO-DHase OMP-PPase OMP-DCase Mutantsin classpyrA H-37 - - + + + 1pyrB M-5; M-8; + _ + + + + 4

M-16; SU-1pyrC G-18; M-3; + + + + + 11

M-4; M-7;M-13; M-14;M-15; M-17;S-3; SU-2;SU-5

pyrD SB319; SB8; + + _ 13GSY289;GSY318; M-2;M-10; M-12;M-18; S-1;S-6; S-7;S-8; MB102

pyrF M-9; SU-7 + + + + + _ 2pyrDHb WB577 + _ + -(+) + + 1pyrDc 17A-42 + + _ _ + + 1pyrDF M-1; S-2 + + + _ + _ 2PYrDBCC 27-6; BR-72; + -(±) -(±) -(+) + + 3

M-11pyrX SB270; SB5; + + + + + + 11

SB305; AU;UTT; A26U;M-6; S-4;168THY -U-;SU-3; SU-8

pyrABC 168TUT _ _ _ + ± + 1pyrACD S-5 + + + 1

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.

I-> 04

0.v) 02D00

FRACTION NO.

FIG. 2. Cosedimentation of three pyrimidine bio-synthetic enzymes in 5 to 20% sucrose density gradi-ents. Abbreviations used: CATase, catalase (beefliver, molecular weight 247,000); CPSase, carbamylphosphate synthetase; ATCase, aspartate transcar-bamylase; DHOase, dihydroorotase. The specific ra-dioactivity of the diluted bicarbonate solution for theCPSase assay was 205 counts/min per nmol.

the pathway as a whole (including CPSase) isschematically presented in Fig. 3. Especiallynoteworthy are the activation of CPSase byornithine and PRPP; the inhibition of the sameenzyme by UTP, dihydroorotate and avidin; theinhibition of DHO-DHase by orotate, carbamylaspartate, guanosine 5'-monophosphate (GMP)and inosine 5'-monophosphate (IMP); and theinhibition of OMP-PPase by UMP, UTP, aden-osine 5'-triphosphate (ATP), and dihydrooroticacid.

(ii) Induction. It was not possible, under ourgrowth conditions, to force wild-type B. subtilisto grow on any of the pathway intermediates.However, a few of the mutants would grow onorotic acid, and extracts from these strainscould be used to ascertain whether any induc-tion of the pyrimidine pathway enzymes hadoccurred. Strain BR72, which is defective inDHO-DHase, was grown with orotic acid andunder uracil-derepressed conditions. Lysozymeextracts were prepared and levels of four en-zymes were compared. Significant increases in

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the specific activities of DHOase and OMP-PPase were observed (1.7-fold and 4.2-fold,respectively); however, we were unable to ob-tain convincing evidence for induction.

(iii) Repression. Strain BK1 shows a drop inactivity for all six enzymes of the pathway whengrown in 5 gg of uracil per ml (Table 2).DHOase, DHO-DHase, and OMP-DCase activ-ities fall to lower levels when the cells are grownin medium containing a 10-fold-higher concen-tration of uracil.Genetic characteristics of the mutants. In

order to expand the linkage map of B. subtilis,the same two strains used in the previousstudies, SB5 and SB319, were used as therecipients in transformation experiments (23).

Competent recipient cells were transformedseparately, with deoxyribonucleic acid fromwild-type cells and from each mutant strain.The calculated recombination indices fromtransformation experiments with strains SB5and SB319 are presented in Table 6. Thelinkage of the CPSasepyr mutation (pyrA) instrain H-37 (Table 1) to the mutations of strainsSB5 and SB319 with co-transformation tech-niques was previouslv reported (34). Only ageneral ordering of groups of similar biochemi-cal mutations, and not an assignment of precisemap positions, is intended.

Correlation of biochemical and geneticdata. The genetic map derived from the trans-formation experiments is represented together

TABLE 5. Inhibition and activation of the pyrimidine biosynthetic enzymes from strain BKI

Relative activitiesEffectora Concn (umol)

ICPSase IATCase DHOase DHO-DHaseb OMP-PPaseb OMP-DCaseI I~~~~~~~10

NoneUMPUMPUTPUTPUTPArg and UTPCTPDihydroorotateDihydroorotateCarbamyl phosphateCarbamyl phosphateCarbamyl phosphateATPATPIMPIMPAMPAMPGMPOrotateOrotateOrotateOMPOMPCarbamyl aspartatePRPPOrnithineArgArgACGLNUridineBiotinAvidin

5105102010 each510

1005

10100

2102105101012.550.55510101010

10010

100101 unitc

1.00

0.58

0.18

0.01

0.690.18

0.45

1.05

3.762.051.090.871.540.720.840.10

1.001.04

1.02

1.070.84

0.91

0.95

0.960.94

1.14

1.05

1.000.80

0.48

0.31

0.76

0.94

0.39

0.57

0.600.72

1.230.901.41

1.00

0.81

0.62

0.84

0.51

0.02

1.580.000.54

0.33

0.700.45

1.00

0.10

0.20

0.47

0.38

0.81

0.62

1.000.87

1.00

0.83

0.95

0.86

0.86

0.89

aAbbreviations used: ACGLN, acetyl-glutamate; Arg, arginine; AMP, adenosine-5'-monophosphate, ATP,adenosine-5'-triphosphate; CTP, cytidine-5'-triphosphate; GMP, guanosine-5'-monophosphate; IMP, ino-sine-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.

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CITRULLINEI-AR61tJlt4E UDPL-ARGININE

SUCCINATE ---.- . UTP_ _:_ - -_

- ARGININE CTP

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

with the enzymatic deficiencies of the mutantsin Fig. 4. It can be seen that most mutants withsimilar enzymatic defects are closely linked onthe map. Unlike other organisms studied previ-ously, in B. subtilis the loci for all pyrimidinebiosynthetic enzymes appear to be contiguous.With the exception of two strains (SB491U5 and168TUT), all mutants with multiple enzymaticdefects lie within the pyrD (DHO-DHase) locus.

DISCUSSIONAll of the pyrimidine auxotrophs (Table 1)

used in this study were derived from B. subtilisstrain "168MI." The lineage and methods ofmutagenesis are clear for all the newly isolatedstrains, but unfortunately this information isdifficult or impossible to obtain for some of theoriginal mutants obtained from other laborato-ries.The results of the transformation experi-

ments with the two recipients, strains SB5 andSB319, varied. The genetic map (Fig. 4) wasgenerally constructed using transformationdata from strain SB5 for those markers fallingin the right third of the map, and data fromstrain SB319 crosses for those in the left two-thirds. It was assumed that the more closely therecipient and donor markers were physicallyassociated, the more accurate the recombina-tion index data were likely to be. The pyrA locusis thought to be located at the extreme left sideof this map on the basis of the positions of themultiple mutations pyrABC (strain 168TUT)and pyrACD (strain SB491U5). Previously re-ported co-transformation data (34) concerningthe CPSasepyr mutation of strain H-37 (Table

1) provide additional support for this assign-ment. The map is merely meant to suggest thelikely order of the groups of mutations andshould not be construed as giving a precise mapposition for each individual mutation. However,it does appear that all the known pyrimidinemutations of B. subtilis fall in an area of thegenome no larger than a single transformationunit. This would correspond to a piece ofdeoxyribonucleic acid having a molecular

TABLE 6. Transformation data

Recombination indexaDonor Recipient Recipient

(SB5) (SB319)

BK1168MIU1168MIU2168MIU3168MIU4168MIU5168MIU6168MIU7168MIU8168MIU9168MIU10168MIU11168MIU12168MIU13168MIU14168MIU15168MIU16168MIU17168MIU18SB491U1SB491U2SB491U3SB491U4SB491U5SB491U6SB491U7SB491U8168SU- 1168SU-2168SU-3168SU-5168SU-7168SU-8GSY289GSY318G18168THY-U -MB45MB46MB102MB106MB169BD71

1.0000.1080.1500.5670.3430.5160.1040.4710.3210.1470.1560.1460.1660.4050.3740.3920.4970.3980.1920.2180.1320.4020.0940.3980.1460.2520.2810.4860.3630.0330.4050.1210.0420.1900.1940.5640.003

0.1750.0000.0000.000

1.0000.0340.0000.4070.2830.2110.1010.3360.2020.0760.0320.0400.0210.3910.4020.3200.2350.3150.0290.0000.0530.4730.2270.4930.1120.1100.0910.2510.3960.1610.2950.1320.2340.1290.0140.2600.3080.1510.1490.007

0.1890.153

aRecombination index was calculated according tothe method of Kelleher and Gooder (23).

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M I M-6SsMM102 SU-7 A

I~ ~ ~ I AU

M-4 GI18 SI M-9 SB305M-15 ~~~~WB577 M-2 S-2 UTTM-15 I7 s- - 8319 M-I SB5

S-5 M-IM4 -1 S-16 - S-7 BS M- -M3 M-5 S fj{jSjjj{jMj1f { } 168THY-u

168TUT SU-2 17A42 76 I S-SS-3 MM5Y8 BR-72 -1 A26U S8270

PYR ABCPYR ACD

'-PYR A

PYR C PYR 8 PYR DPYR DBPYR DCPYR DSPYR DX

PYR

PYRDF

PYR X

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; andstrains designated "SU-" are from the 168SU series. The letters following "pyr" correspond to deficientenzymes 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% ofthe entire genome (2, 11).

In the cases of the two possibly rate-limitingsteps of the pathway, CPSase and OMP-PPase,the enzymatic activities were reproducible al-though usually quite low. The strains that havebeen found to lack detectable pyrimidine-specific CPSase activity (SB491U5, H-37, and168TUT) have been retested at least 10 times.The S. typhimurium CPSase deletion mutant(pyrA81) has been observed to yield quantita-tive results that are very similar to those ob-served for the B. subtilis mutants mentionedabove. It is not possible to determine thesensitivity of this assay procedure, unless par-tially purified carbamyl phosphate synthetasebecomes available.

Previous work in this laboratory has demon-strated that the OMP-PPase activity from B.subtilis deviated from linearity within one tothree minutes. The possibility that this problemmight be a result of feedback inhibition byOMP, the product of the enzyme (reported forE. coli B by Lieberman and Simms [25]), is notsubstantiated by our observation that the addi-tion of purified yeast OMP-DCase does notprolong the B. subtilis OMP-PPase linear activ-ity. An attractive possibility would be that somefactor, either a membrane fragment or somehigh-molecular-weight component normally as-sociated with the membrane, is undergoing aseparation from the enzyme involving centrifu-gation-induced changes in enzyme conforma-tion such as might be inferred from the in-creased period of linearity observed when crudeextracts were centrifuged for 60 min instead of30 min (see Results).During her work with the membrane-bound

E. coli DHO-DHase, Karibian (20) discoveredthat Triton X-100 could restore DHO-DHaseactivity that had been destroyed by treatmentwith phospholipase A2. In E. coli, BRIJ-35,

Triton X-100, and bovine serum albumin canall substitute to some extent for lost lipids torestore enzymatic activity (20, 21). Our obser-vations concerning the sedimentation behaviorof some of the pyrimidine biosynthetic enzymesin B. subtilis may be viewed as suggestiveevidence of a particulate association of theseenzymes. Further evidence for possible mem-brane attachment was obtained from studies onCPSase (34).The results of the sucrose density gradient

demonstrated that CPSase, ATCase, and DHO-ase exhibited maximal activity at precisely thesame location in the gradient (Fig. 2). Similarresults had previously been obtained in mam-malian cells (38) and in Serratia marcescens(Wild, Ph.D. dissertation, University of Califor-nia, Riverside, 1972). In both of these cases, anenzyme complex is thought to exist. In mamma-lian cells, the CPSase-ATCase-DHOase com-plex has a molecular weight of approximately750,000 to 850,000, whereas the last two en-zymes, OMP-PPase and OMP-DCase, form asecond aggregate with a molecular weight of100,000 to 140,000 (Fed. Proc. 31:473, 1972).The results reported here for the first threepyrimidine enzymes of B. subtilis indicate amolecular 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 previ-ously described in other organisms. Six of theothers, composed of eight mutants, displaymultiple enzymatic defects under our in vitroassay conditions. The genetic evidence showsthat six of these strains contain mutationswhich are located within the pyrD (DHO-DHase) locus (Fig. 4), and the biochemical dataindicating unstable activity for some of thedefective enzymes (Table 3) led to speculationconcerning the nature of these mutations. Anexamination of the various combinations of

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enzymatic defects rules out classical polarityeff'ects. The observed reversion frequencies of10- to 10-9 for these same six strains providenegative evidence for deletions or multiple mu-tations. Reversion of the other two mutants,strains 168TUT and SB491U5 (Table 1), whichlie some distance from the pyrD locus, has notbeen observed. The most likely explanations forthese multiple defects would seem to be thateither a single mutation is altering the confor-mation and activities of' more than one enzyme,or a defect in a single enzyme produces apleiotropic effect on other enzymes throughsome complex regulatory mechanism. Note,however, that mutants with a defective DHO-DHase do not always have other enzymaticdeficiencies.One of the most interesting classes of mu-

tants, pyrX, includes seven strains. All at-tempts to demonstrate an enzymatic defectwith in vitro methods have been unsuccessful.The massive excretion of orotate by most pyrXmutants, along with the other observationsmentioned in the Results section, leads one toconclude that pyrX mutants may lack in vivo(and possible in vitro) activity for either OMP-PPase or OMP-DCase. On the basis of thelocations of their mutations (directly adjacentto the OMP-DCase locus) and the absence ofany known B. subtilis OMP-PPase mutants, itis possible that they may be leaky pyrE mu-tants. Additional support for this theory wasrecently obtained when three of' the pyrX mu-tants (strains A26U, SB5, and SU-8) wereretested for OMP-PPase activity by using aslight modification of the method of Shoaf andJones (38) and were found to have less than 4%of the activity of wild-type strain BK1. Thepossibility that pyrX mutants may have defec-tive pyrimidine-specific PRPP synthetase activ-ity can also not be ruled out.

Unlike the enzyme of most enteric bacteria,the B. subtilis ATCase is insensitive to feedbackcontrol. The CPSase is, however, subject toboth feedback inhibition and activation (Fig.3). The regulatory consequences of these prop-erties as well as the interaction of CPSase withproteins such as avidin and lysozyme have beenpreviously discussed (34). It is interesting tonote that UTP has a moderately inhibitoryeffect on DHOase in addition to its muchstronger action on CPSase. This could arise dueto the multi-enzyme complex suggested by thesucrose density gradient work. Along the sameline of reasoning, CPSase is somewhat (-5O0%)inhibited by the product of DHOase. The othermajor sites of inhibition in the pyrimidine

pathway are DHO-DHase (by orotate) andOMP-PPase (by UMP).One f'inal point is pertinent. In B. subtilis, the

balanced (steady-state) production of purinesand pyrimidines is partially accomplished byPRPP concentration. PRPP is used as a sub-strate by both the purine and pyrimidine path-ways, is a powerf'ul activator of CPSase (34),and inhibits adenosine-5'-monophosphate syn-thetase (17). In S. typhimurium, the enzymethat synthesizes PRPP. phosphoribosylpyro-phosphate synthetase, is repressed by uridinecompounds (33). Additional control of the en-zyme is provided in B. subtilis by IMP andGMP, which strongly inhibit DHO-DHase (Fig.3; Table 5).On the basis of the information gathered

during this study, a multienzyme complex inwhich DHO-DHase is the central enzyme issuspected to exist in B. subtilis. The five otherenzymes are most likely associated with thecentral enzyme in two groups: CPSase-ATCase-DHOase and OMP-PPase-OMP-DCase. A simi-lar complex was previously proposed for B.subtilis by Kelleher (Ph.D. dissertation, Uni-versity of North Carolina, Chapel Hill, 1969)and for mammalian cells by Shoaf and Jones(M. E. Jones, personal communication). Thismodel seems to fit most of the available data,and makes it possible to visualize how a muta-tional alteration in the configuration of theDHO-DHase enzyme could cause a loss ofactivity in anv of the five other enzymes.

ACKNOWLEDGMENTS

We wish to thank Kenneth Bott and Robert Twarog whogenerously provided the use of their equipment, expertise,and advice, and Gerard O'Donovan for his many helpfulsuggestions and his critical reading of this manuscript.

This investigation was supported by a United MedicalFoundation grant 324UNI (256) and also by grant AI-04577from the National Institute of Allergy and Infectious Dis-eases. B. W. P. and R. J. K. were supported by NationalInstitute of General Medical Services grant GM006-85.

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38. Shoaf, W. T., and M. E. Jones. 1973. Uridylic acidsynthesis in Ehrlich ascites carcinoma. Properties,subcellular distribution, and nature of enzyme com-plexes of the six biosynthetic enzymes. Biochemistry12:4039-4051.

39. Taylor, A. L., and C. D. Trotter. 1972. Linkage map ofEscherichia coli strain K12. Bacteriol. Rev. 36:504-524.

40. Waindle, L., and R. L. Switzer. 1973. Inactivation ofaspartic transcarbamylase in sporulating Bacillussubtilis: demonstration of a requirement for metabolicenergy. J. Bacteriol. 114:517-527.

41. Yan, Y., and M. Demerec. 1965. Genetic analysis ofpyrimidine mutants of Salmonella typhimurium. Ge-netics 52:643-651.

42. Young, F. E., and G. A. Wilson. 1972. Genetics of Bacillussubtilis and other gram-positive sporulating bacilli, p.77-106. In H. 0. Halvorson, R. Hanson, and L. L.Campbell (ed.), Spores V. American Society for Micro-biology, Washington, D. C.

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ERRATA

Pyrimidine Biosynthetic Pathway of Bacillus subtilisBARRY W. POTVIN,* RAYMOND J. KELLEHER, JR., AND HARRY GOODER

Curriculum in Genetics and Department of Bacteriology and Immunology, School of Medicine, University ofNorth Carolina, Chapel Hill, North Carolina 27514

Volume 123, no. 2, p. 604, column 1, lines 19-24: Should read .. . A major site of regulation in thepyrimidine pathway of many bacteria seems to be aspartate transcarbamylase (ATCase, EC 2.1.3.2)(13). In Salmonella typhimurium and E. coli, cytidine-5'-triphosphate (CTP) is the feedbackinhibitor . . ."Page 604, column 2, line 16: "L. J. Rebello" should read "J. L. Rebello."

Genetic Analysis of Thymidine-Resistant and Low-Thymine-Requiring Mutants of Escherichia coli K-12 Induced by

Bacteriophage Mu-1R. S. BUXTON

Division of Microbiology, National Institute for Medical Research, Mill Hill, London NW7 1AA, England

Volume 121, no. 2, p. 477, Table 2, column 11: "20,ug/ml" should read "0 ug/ml."Page 479, legend to Fig. 2, line 6: "thyAC-" should read "thyA-."

595

pyrimidine biosynthetic pathway bacillus subtilis · 2006. 3. 10. · pyrimidine biosynthetic...

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