JOURNAL OF BACTERIOLOGY, Nov. 1968, p. 1732-1741 Vol. 96, No. 5Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Control of Pyrimidine Biosynthesis inPseudomonas aeruginosaJILLIAN H. ISAAC1 AND B. W. HOLLOWAY'

Faculty ofScience, Monash University, Clayton 3168 and School of Microbiology,University ofMelbourne, Parkville 3052, Victoria, Australia

Received for publication 12 August 1968

The pathway of pyrimidine biosynthesis in Pseudomonas aeruginosa has beenshown to be the same as in other bacteria. Twenty-seven mutants requiring uracilfor growth were isolated and the mutant lesions were identified. Mutants lackingeither dihydroorotic acid dehydrogenase, orotidine monophosphate pyrophos-phorylase, orotidine monophosphate decarboxylase, or aspartic transcarbamylasewere isolated; none lacking dihydroorotase were found. By using transduction andconjugation, four genes affecting pyrimidine biosynthetic enzymes have beenidentified and shown to be unlinked to each other. The linkage of pyrB to met-28and ilv-2 was shown by cotransduction. Repression by uracil alone or by brothcould not be demonstrated for any enzymes of this pathway, in contrast to thesituation in Escherichia coli and Serratia marcescens. In addition, derepression ofthese enzymes could not be demonstrated. A low level of feedback inhibition ofaspartic transcarbamylase was found to occur. It is suggested that the control ofsuch constitutive biosynthetic enzymes in P. aeruginosa may be related to thecomprehensive metabolic activities of this organism.

It is known that the arrangement of genes inbacteria can have a significance for the control ofgene products. In particular, the operon conceptrelates the contiguous arrangement of structuralgenes to their transcription and control by regu-latory genes. This contiguous arrangement is byno means universal, and both clustered and un-clustered arrangements are found in Escherichiacoli. Already, 10 operons have been mapped inE. coli (18), and undoubtedly more remain to beidentified.Such an arrangement does not seem to be

universal among bacteria, and variations on thistheme are important to our complete under-standing of enzyme control mechanisms. Themetabolic behavior of Pseudomonas is differentin a variety of ways from that of E. coli. Further-more, mapping of Pseudomonas has not revealedto date any evidence of the gene clustering neces-sary for the typical operon structure, and, whenthe same pathways have been mapped in bothbacteria, the gene arrangement in P. aeruginosahas been found to be quite different. In no caseexamined to date is there clustering of all thegenes of any one pathway. Instead, there appearto be aggregations of two or three genes, but these

1 Present address: Faculty of Science, Monash Uni-versity, Clayton 3186 Victoria, Australia.

aggregations are not linked to each other andappear to be widely distributed over the chromo-some.

In three of the studied pathways, there appearsto be a mixture of clustered and unclustered genes[isoleucine plus valine (15), tryptophan (6), andhistidine (13)]. Pathways in which all the genesare contiguous and have been shown to have anoperon structure in E. coli, e.g., leucine or tryp-tophan, clearly have a different gene arrangementin P. aeruginosa.

Pseudomonas is known to possess synthetic anddegradative abilities not found in E. coli. Theability to use a wide variety of substrates as car-bon and nitrogen sources (17) is well-documented,and a clearer understanding of the genetic basisof these properties is needed.The different arrangement of genes on the

chromosome immediately raises the questionwhether the genetic regulation of biosynthesis andmetabolism is the same in P. aeruginosa as inother bacteria. Crawford and Gunsalus (3) haveshown for P. putida that, unlike E. coli, not allthe sequential steps in the biosynthesis of trypto-phan are regulated by end-product repression. Acombined genetic and enzymatic analysis (2) hasshown that there are three linked loci controllinganthranilate synthetase, phosphoribosyl trans-

1732

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

PYRIMIDINE BIOSYNTHESIS IN P. AERUGINOSA

ferase, and indoleglycerol phosphate synthetase.These have all shown coordinate repression bytryptophan. The locus for phosphoribosyl an-thranilic isomerase (PRAI) is unlinked to any ofthese three, or to the two linked loci for the Aand B subunits of tryptophan synthase. PRAIdoes not show end-product repression and tryp-tophan synthase A and tryptophan synthase Bshow control by substrate induction. The furtherunderstanding of these different patterns of ge-netic control can only come from the detailedstudy of a variety of pathways in several or-ganisms.We have selected the pyrimidine pathway to

further examine the question of the relation ofgene distribution and control mechanism in P.aeruginosa. It has been previously shown that allsix of the enzymes in the pyrimidine pathway ofE. coli are subject to regulation (1). Four of theenzymes involved, dihydroorotase, dihydrooroticacid dehydrogenase, orotidine monophosphatepyrophosphorylase, and orotidine monophos-phate decarboxylase, were shown to be coor-dinately repressed. Initially, all four genes con-trolling these enzymes were thought to be closelylinked, but further work has shown that onlytwo, pyrC dihydroorotase and pyrD dihydrooroticacid dehydrogenase, are apparently contiguous,the remaining genes all being unlinked to eachother or to pyrC or pyrD (18).

MATERIALS AND MErHoDs

Bacteria. All mutant strains were derived from P.aeruginosa PAO1. The list of uracil-requiring mu-tants examined is shown in Table 1, together withother strains used in this work.

Bacteriophage. Transduction studies were madewith F116 (6). The virulent phage E79 was used inthe interrupted mating technique, by using the tech-nique previously described (19).

Media. Minimal medium (MM) was that of Vogeland Bonner (20) and contained 0.20% glucose,0.20% citric acid, 1% K2HPO4 anhydrous, 0.35%NaNH4HPO4-4H20, and 0.02% MgSO4-7H20. MMagar plates consisted of MM solidified with 1.8%agar (Difco). Nutrient yeast broth contained 0.8%Nutrient Broth (Difco) plus 0.5% Yeast Extract(Difco). Meat infusion agar contained 0.1% beefextract (Bonox), 1% peptone (Evans), 0.8% sodiumchloride, and 1.2% agar (Davis).

Chemicals. N-methyl-nitro-N-nitrosoguanidine wasfrom Aldrich Chemical Co., Inc., Milwaukee, Wisc.;carbamyl phosphate (dilithium salt), L-asparticacid, and uracil were from Calbiochem, Los Angeles,Calif. Carbamyl aspartic acid, dihydroorotic acid,orotic acid, phosphoribosyl 5' pyrophosphate, anduridine monophosphate were from Nutritional Bio-chemicals Corp., Cleveland, Ohio.

Sodium diphenylamine p-sulphonate and potas-sium persulphate were from British Drug Houses.

TABLE 1. Bacterial strains used in this studya

Strain no.

PAOI

PAO109PA0116PA0129PA0145PA0146PA0180

PA0113PA0114PA0117PAO119PA0138PA0150PA0151PA0291PA0484

PA0483

PAO103PAOlW4PA0105PA0106PA0141PA0148PA0160PA0161PA0162PA0163PA0164PA0165

PA08

PA02

Genesymbol

pyrB

pyrD

pyrE

pyrF

met-28ilv-2

ser-3

Description

Wild-type parent,prototrophicstrain 1.

Aspartate trans-carbamylase-deficient

Dihydrooroticacid dehy-drogenase-de-ficient

Orotidylic acidpyrophospho-rylase-de-cient

Orotidylic aciddecarboxylase-deficient

Methionine-re-quiring, iso-leucine- andvaline-requir-ing

Serine-requiring

Reference

(10)

This study

This study

This study

This study

In pressb

(11)a Where possible, we have used the same sym-

bols as Taylor and Trotter (18) and have followedthe conventions suggested by Demerec et al. (4)for genotypic and phenotypic symbols.

bB. Rolfe and B. W. Holloway, Genetics, inpress.

Isolation of mutants. Exponentially growing cellsof P. aeruginosa PAO1 were treated with 20 yg ofN-methyl-nitro-N-nitrosoguanidine per ml in citratebuffer (pH 5.0) at 37 C for 20 min. The cells werewashed twice in isotonic saline, inoculated intonutrient yeast broth, and shaken at 37 C for 4 to 6hr to allow expression of any mutations. Samples of

VOL. 96, 1968 1733

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ISAAC AND HOLLOWAY

a dilution to give between 100 and 200 colonies perplate were then spread on MM agar plates enrichedwith 1.25% nutrient yeast broth. After 48 hr of incu-bation, any small, faint colonies were picked off andtested for growth on MM and MM supplementedwith 20 Ag of uracil per ml. Such faint colonies ap-peared at a frequency of approximately 0.5 to 1%;the frequency of auxotrophic mutants isolated fromthese faint colonies was about 30%, and the fre-quency of uracil auxotrophs was 1 to 2% of totalauxotrophs. To prevent the isolation of sibling mu-tants, only one mutant of each type was selectedfrom each mutant isolation run.

Preparation of extracts for enzyme analysis. Cellswere grown aerated at 37 C in liquid MM or liquidMM supplemented with 1-83M uracil (112 ug/ml)until midexponential phase (OD 680 nrm = 0.6). Thecells were cooled in ice, spun at 13,000 X g for 20min at 4 C, resuspended in 4 ml of 0.5 M tris(hy-droxymethyl)aminomethane, (Tris) pH 7.8, per g(wet weight) of cells, and smashed at 20,000 lbs/in2with a French press. Cellular debris was not removedby centrifugation, as it has been reported that thedihydroorotic acid dehydrogenase, one of the en-zymes under investigation, is membrane-bound (19).Enzyme assays. The incubation mixture for aspar-

tic transcarbamylase assay contained (in 0.5 ml): 100Amoles of Tris (pH 8.8), 10 pnoles of L-aspartic acidneutralized to pH 8.8 by sodium hydroxide, and 10umoles of carbamyl phosphate (dilithium salt).After equilibration at 28 C for 5 min, the reactionwas commenced by the addition of enzyme extractcontaining approximately 0.2 mg of protein. After 10min of incubation at 28 C, the reaction was stoppedby the addition of a reagent described by Yates andPardee (22). Development of the color response tocarbamyl aspartic acid was carried out as describedby Yates and Pardee, except that the color wasallowed to develop at room temperature and theoptical density was read 30 min after the addition ofpotassium persulphate (OD 560 nm = 0.36 for 0.1,umole of carbamyl aspartic acid).

The assay for dihydroorotase was carried out asdescribed by Beckwith et al. (1), except that theincubation period was 15 min with 0.2 mg of protein.The assay for dihydroorotic acid dehydrogenase was

carried out as described by Beckwith et al. (1). Theassay for orotidine monophosphate pyrophosphoryl-ase was carried out as described by Beckwith et al. (1),except that only 0.1 iAmole of orotic acid was added,this concentration being saturating for 0.03 mg ofprotein. Assay for orotidine monophosphate de-carboxylase could not be carried out, as orotidinemonophosphate was unavailable.

Protein determinations. The protein content of theextracts was determined by the Folin method (12)with bovine serum albumin (fraction V) as thestandard.

Unless otherwise stated, all enzyme activities weremeasured at saturating substrate concentrations andunder conditions such that the initial velocity of thereaction was being measured.

RESULTSEstablishment of the pyrimidine biosynthetic

pathway in P. aeruginosa. A series of uracil-requiring mutants were isolated and tested fortheir ability to grow on intermediates of thepyrimidine biosynthetic pathway as establishedin E. coli (1) and Serratia marcescens (9 andFig. 1). Each mutant was suspended in turn in anagar layer, crystals of the suspected intermediateswere placed on sterile filter paper discs, and theplates were examined for zones of growth after48 hr of incubation. In all cases, luxuriant growthwas observed around uracil, but no growthresponses to the possible intermediates of car-bamyl phosphate, aspartic acid, carbamyl aspar-tate, dihydroorotic acid, orotic acid, or uridine5' monophosphate were observed. To enhancethe permeability of the cell to the acidic inter-mediates, the cells were also incubated in liquidmedium at pH 5.0 with a concentration of 10-3M of each of the intermediates; again, no growthwas observed after 48 hr of incubation.The lack of growth on suspected intermediates

could be explained by either low permeability ofthe cell for the substances tested or by the fact

carbamynll phosphate AT c1.DosHO'.crmlPOhtAC'scarbamyl L-aspartic D.H0a* L-dihydro orotic

;t- 4 acid acid

L-aspartic acid

DRO.d.h'ase

uridine i4Ot Pd.c'n orotidine O.M.P. '. L-orotic acid5'monophosphate ' monophosphate

P.tpFIG. 1. Pathway of pyrimidine biosynthesis. Abbreviations: ATC'ase, aspartic transcarbamylase; DHO'ase,

dihydroorotase; DHOdeh'ase, dihydoorotic acid dehydrogenase; OMPdec'ase, orotidine monophosphate decar-boxylase; OMPpp'ase, orotidine monophosphate pyrophosphorylase.

1734 J. BACTERIOL.

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

PYRIMIDINE BIOSYNTHESIS IN P. AERUGINOSA

that the suspected intermediates are not theintermediates of the pyrimidine pathway in thisorganism.To determine whether the pathway was the

same as in E. coli and S. marcescens, cell-freeextracts of the wild type grown in liquid MMwere examined for activity of the suspected en-zymes. Under the assay conditions describedabove, activity of the enzymes aspartic transcar-bamylase, dihydroorotase, dihydroorotic aciddehydrogenase, and orotidine monophosphatepyrophosphorylase was demonstrated (Table 2).

It thus appears that, although no positiveidentification of the enzyme orotidine monophos-phate decarboxylase has been made (because ofthe unavailability of the substrate), it is reasonableto conclude that the pathway of uracil biosyn-thesis in P. aeruginosa is essentially the same asthat of E. coli and S. marcescens.To identify the particular enzyme deficiency in

the uracil-requiring mutants isolated, assays for

TABLE 2. Activities of pyrimidine biosyntheticenzymes in P. aeruginosa

Enzyme Activitya pH ofassay

Aspartic transcarbamylase 4.5 0.4 8.8Dihydroorotase 1.0 :1 0.2 8.6Dihydroorotic acid dehy-drogenase 0.24 0.02 8.4

Orotidine monophosphatepyrophosphorylase 0.60 i4 0.05 8.8

a Expressed as micromoles of product formedper hour per milligram of protein.

each of these four enzymes were made with eachof the 28 different mutants (Table 3).

It had previously been shown, in an overallstudy of gene linkage relationship in biosyntheticpathways of P. aeruginosa (6), that there is littleclustering of genes controlling sequential bio-synthetic steps, and in no case were all the genescontrolling one pathway arranged contiguously.We have carried out a similar analysis on the

uracil-requiring mutants isolated for this study(strain 1, PAO 1, was used instead of strain 2,PAT 2, which was used in the previous work).We found that there is no clustering of pyrimidinemarkers and all four genes identified are unlinkedto each other.Two types of transduction analyses were car-

ried out. In the first, each uracil-requiring mutantwas used in turn as donor and recipient fortransduction with phage F116; the frequency offormation of prototrophic transductants com-pared with that obtained with phage propagatedon a wild-type donor. If any of the uracil mutantshave closely linked lesions, the frequency ofprototrophs will be reduced. Likewise, if thefrequency is found not to be reduced, it is sub-stantial evidence that the genes concerned are notlinked. From Table 4, it is seen that there is nolinkage by this test between any of the four genesof the uracil pathway.Although this test confirms the homogeneity of

the groups, it does not absolutely confirm thatthere is nonlinkage. Further linkage studies werecarried out by using the donor phenotype selec-tion test previously used (6).Phage F116 was propagated on a selected mu-

TABLE 3. Identification ofenzyme lesion in uracil-requiring mutants

Mutant no. ATC'ase DHO'ase DHOdeh'- OMPpp'- Lesionase ase

PAO109, PAO116 - + + + ATC'asePA0129, PA0145PA0146, PAO180

PAO113, PAO114, PAO117 + + - + DHOdeh'asePAO119, PA0138, PAO150PAO151, PA0291, PA0484

PA0483 + + + _ OMPpp'ase

PAO103, PAO104 + + + + Presumably OMPdec'asePAO105, PAO106PAO141, PA0148PAO160, PAO161PA0162, PA0163PA0164, PA0165

a Enzyme present, +; no enzyme detected,-.

VoL. 96, 1968 1735

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ISAAC AND HOLLOWAY

TABLE 4. Prototroph numbers obtained in transductions between pyr mutants of different groups with phageF116

Donor

Recipient

PA0129PAO180PAO113PAO119PA0483PAO106PAO161PA0162PAO2

ATC'ase DHOdeh'ase OMPpp'ase OMPdec'ase Nild type

PA0129

Oa2

1849060

422336488337

PAO180

30

1021212985180220184

PAOI 19

150400

0

23321227206292

PAOl51

1353623

39360208191204

PA0483

28733

178880

403598430483

PAO141

14028604560

0

0

137

PAO161

27352

22787830

0

0

409

PAO162

135759780300

0

0

408

PAOI

9860908532

376240419258

a Each figure represents the average obtained on three minimal plates after adjustment for backmutation. An overnight broth culture (2 to 4 X 109 bacteria per ml) was infected with F116 at a mul-tiplicity of between 2 and 4. After 15 min of adsorption at 37 C, the infected culture was centrifuged,resuspended in saline, and 0.1 ml was plated onto each of three minimal plates.

tant from each group and was used as a donor forthe selection of prototrophic transductants froma range of different auxotrophs. The two trans-ductants were selected on minimal medium sup-plemented with uracil, so that, if any linkage ofthe uracil marker and any other markers occurred,selection for the prototrophic allele of the re-

cipient marker would involve selection for thepyr allele and enable its survival on MM anduracil medium.The distribution of genes in P. aeruginosa

makes this somewhat haphazard method of map-ping more likely to succeed, as the nonclusteringof related pathways means that there are morepotential sites for such transduction linkage thanis the case with, e.g., E. coli, where clusteringcommonly occurs. This method has already beenfound to be successful for the location of a

fluorophenylalanine-resistance marker site (21).More than 30 different auxotrophic mutants

were used as acceptors, and linkage between twoof these and one of the uracil sites has been shown(Table 5). With the aspartic transcarbamylasemutants (pyrB) PA0129 and PAO180, cotrans-duction is seen with the markers met-28 andilv-2, already known to be cotransducible (17).The data from Table 5 show that the order ofthese three markers must be pyrB met-28 ilv-2.

Attempts were made to map the remainingthree pyr markers in P. aeruginosa by interruptedmating (V. A. Stanisich and B. W. Holloway, inpress). All three markers have low frequencies ofrecombination in conjugation and all apparentlyenter late (after at least 70 min). Owing to thenature of this conjugation system, mapping ofmarkers which enter as late as this is unreliable

TABLE 5. Cotransduction of pyrimidine genes withother markers by phage F116G

Donor Met- Met+ iMet- Met+ Ilv- Ilv+ Ilv- Ilv+Ura- Ura- Ura+ Ura+ Ura7 Ura7 Ura+ Ura+

PA0129 0 5 112 28 63 3 111 29PAO180 0 6 142 24 54 4 82 19PA0483 0 0 145 37 0 0 75 66PA0162 0 0 97 23 0 0 81 70PAO119 0 0 110 18 0 0 70 44

a Phage F116 was grown on each uracil mutantin turn. The double mutant met-28, ilv-2 (PAO8)was used as recipient; after phage infection, thecells were plated on uracil plus methionine MM-agar and uracil plus isoleucine plus valine MM-agar. Individual colonies were then picked andreplica-plated to determine their phenotype. Thenumbers represent the number of colonies havingthe respective phenotype shown.

with existing techniques, and no precise times ofentry or linkage relationships could be demon-strated.

Because the enzyme lesion for orotidine mono-phosphate decarboxylase could not be identifiedowing to unavailability of the substrate, it ispossible that this group of mutants could beheterogeneous. There is no evidence for this,because, whereas all the mutants in the grouphave not been tested by transduction analysisagainst all the other classes of uracil mutants,transduction tests by the prototroph selectionmethod within the group of orotidine monophos-phate decarboxylase mutants (listed in Table 1under pyrF) show no evidence of genetic hetero-geneity of this group.

1736 J. BACTERIOL.

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

PYRIMIDINE BIOSYNTHESIS IN P. AERUGINOSA

From these results, it can be concluded thatthe four uracil markers identified in P. aeruginosaare not clustered or contiguous and are notclosely linked to each other.

In both E. coli and S. marcescens, it has beenshown that control of biosynthesis of enzymes inthis pathway is by repression and that the unusualsituation of coordinate repression of unlinkedgenes occurred, at least in E. coli. In view of thedifferent pattern of gene distribution in P. aeru-

ginosa, the enzyme control mechanisms werestudied.

Repression. It has been demonstrated for manybiosynthetic pathways in a variety of organismsthat the rate of synthesis of the enzymes involvedin the biosynthesis of a compound is decreasedmarkedly by the presence in the growth mediumof the particular compound, or by growth in ageneral growth medium such as broth.With E. coli (AB259) cells grown in nutrient

broth, harvested, and inoculated into a minimal(glucose-salts-Bl) medium, there is a lag periodof up to 10 hr before cell division commences.With P. aeruginosa, in a similar transfer of cellsfrom an enriched to an impoverished medium,the lag period is only 80 to 90 min (i.e., about 1.5generations, minimal). Such a result suggeststhat the general pattern of repression in P. aeru-

ginosa may be quite different from that in E. coli.We thus looked at the nature of repression of

uracil-synthesizing enzymes in P. aeruginosa. Thelevels of the pyrimidine biosynthetic enzymes inexponentially growing cells of P. aeruginosagrown in minimal medium, minimal mediummade 103 M with respect to uracil, and in nutrientyeast broth, were compared (Table 6); there was

no evidence of repression. A similar lack ofrepression for enzymes involved in the biosyn-thesis of phenylalanine has also been shown forP. aeruginosa (J. A. Waltho, Ph.D. Thesis, Univ.of Melbourne, 1968).The results recorded in Table 6 differ from

those demonstrated in E. coli by Beckwith et al.(1), who showed that growth in minimal mediumcontaining 9 X 10-4 M uracil caused a sixfolddecrease in the level of aspartic transcarbamylase,

and twofold decreases in the levels of dihydro-orotase, dihydroorotic acid dehydrogenase, oro-tidine monophosphate pyrophosphorylase, andorotidine monophosphate decarboxylase, whencompared to growth in minimal medium alone.We repeated such experiments in our laboratorywith E. coli K-12 strain AB259, with the same

techniques for growing the cells, preparing thecell-free extract, and assaying the enzymes that weused for P. aeruginosa. We found decreases inenzyme levels similar to those reported by Beck-with et al. (1). This indicates that, if a system ofrepression had occurred in P. aeruginosa similarto that known to occur in E. coli K-12, then itwould have been demonstrable by the techniquesused.One possible complication in studies of this

type in P. aeruginosa is that, in the present case,uracil is being degraded and used for growthpurposes. We found that P. aeruginosa PAO1can grow on a medium containing uracil atsaturating concentrations (25 mM), but the lagperiod is greater than 48 hr and the generationtime is about 6 hr. We thus consider that, in theexperiments reported here on the pattern ofenzyme control of uracil in P. aeruginosa, thedegradation of uracil is unlikely to be a sig-nificant factor.We can suggest three possible explanations for

the inability to demonstrate repression: (i) uracilfrom the growth medium not being taken up intothe cell; (ii) intracellular level of uracil in thewild-type cell grown in minimal medium is suf-ficient to effect repression of pyrimidine enzymesin absence of exogenous uracil, therefore, addeduracil has no effect; (iii) repression is not a majormechanism of control for this organism in thispathway.

Uracil uptake. Pyr- mutants respond very wellto added uracil, indicating that the cell is perme-able to uracil, although it is possible that thelevel of uracil in the wild type may be too highfor uracil uptake to occur. However, van de Putte(personal communication) has shown that the rateof uptake of 14C-uracil into wild-type cells of P.aeruginosa PAO1 is similar to that of E. coli K-12.

TABLE 6. Levels ofuracil biosynthetic enzymes in P. aeruginosa grown in the presence and absence ofuracil

Enzyme activity4Medium

ATC'ase DHO'ase DHOdeh'ase OMPpp'ase

Minimal.4.2 i 0.2 1.0 4 0.1 0.25 4+ 0.03 0.59 + 0.03Minimal + 103 M uracil 4.1 :1: 0.2 1.0 + 0.1 0.26 + 0.03 0.60 +t 0.03Nutrient yeast broth 4.4 4± 0.2 1.2 4 0.1 0.24 i 0.03 0.58 ± 0.03

Expressed as micromoles of product formed per hour per milligram of protein. Results are pooledfrom a number of independent determinations.

VOL. 96, 1968 1737

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ISAAC AND HOLLOWAY

For these reasons, we have concluded that poor

uracil uptake is not the reason for inability todemonstrate repression.

Intracellular pyrimidine. There are several waysof limiting the intracellular level of pyrimidines.The intracellular level of uracil and uracil deriva-tives in a pyrimidine-requiring mutant must bevery low or zero. According to the model ofrepression, if uracil or a derivative of uracilrepresses the formation of pyrimidine enzymes,then, in a uracil mutant, the system should bederepressed and the level of pyrimidine biosyn-thetic enzymes should rise when the externalsupply of uracil has been exhausted.

Activities of the various enzymes at differenttimes after the external uracil supply has beenexhausted were examined in three different py-rimidine-requiring mutants. Replicate cultureswere grown in limiting uracil and were harvestedat the same cell density (within 5%) at differenttimes after exhaustion of uracil. Enzyme levelswere compared with those of cells grown inexcess uracil and harvested at the same celldensity. Cells were always harvested at a celldensity corresponding to mid-exponential phaseof the strain when grown under nonlimitingconditions.The results (Table 7) clearly show that there is

no derepression of enzyme synthesis under condi-tions of limiting uracil. This inability to demon-strate derepression invalidates any hypothesiswhich suggests that the endogenous repression iscomplete in wild-type cells, owing to high poolconcentration of uracil, and thus addition ofexogenous uracil is without effect.

It is possible that the failure to demonstratederepression in the above experiments may bedue to inability of the system to synthesize pro-tein under the conditions used. This objection can

be overcome by using a bradytrophic derivativeof a uracil mutant which shows slow growth onminimal medium, and thus provides the equiva-lent of an "internal" chemostat in which uracilis the growth-limiting factor.We isolated such bradytrophic mutants by

ethyl methane sulphonate treatment (7) ofPAOI 19, followed by plating on minimal mediumand the isolation of slow-growing colonies.

In one such mutant, the generation time inminimal medium plus 10-3 M uracil was 55 min(the same as wild type in minimal medium), ascompared to 140 min when grown in minimalmedium without uracil. When the enzyme levels ofsuch a bradytrophic mutant growing in minimalmedium plus excess uracil were compared withthe levels of the same strain growing in minimalmedium, no difference was found, showing thateven when growth, and hence protein synthesis,could take place, no derepression of uracil bi-osynthetic enzymes occurred.Growth of the wild type in a substance which

inhibits pyrimidine synthesis should result in lowintracellular levels of uracil. 6-Azauracil is auracil analogue and a competitive inhibitor oforotidine monophosphate decarboxylase (8). Bygrowing the wild type in a concentration of6-azauracil which is just sufficient to inhibitgrowth, the intracellular level of uracil can beassumed to be low and growth-limiting.There are two possible reasons why growth in

6-azauracil might result in an increase in enzymelevels. First, the inhibition of orotidine mono-phosphate decarboxylase causes the wild type tobehave like a mutant, and derepression mayoccur. Second, the accumulation of substratescaused by the inhibition of orotidine monophos-phate decarboxylase may result in induction bya substrate of one or all of the enzymes.

TABLE 7. Enzyme levels of uracil-requiring mutants growing in limiting quantities of uracil

Time of Enzyme activityaMutant deprivation

(min) ATC'ase DHO'ase DHOdeh'ase OMPpp'ase

PAO113 (DHOdeh'ase) 0 4.1 1.1 0.5630 3.9 1.2 0.5960 3.9 0.9 0.5690 4.2 1.0 0.55120 4.2 1.1 0.59

PAO103 (OMPdec'ase-) 0 4.3 0.7 0.41 0.7015 4.4 0.9 0.32 0.7225 4.3 1.0 0.40 0.6730 4.2 1.0 0.34 0.75

PA0483 (OMPpp'ase-) 0 4.5 0.9 0.3130 4.6 1.0 0.2460 5.0 1.0 0.25

a Expressed as micromoles of product formed per hour per milligram of protein.

1738 J. BACTERIOL.

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

PYRIMIDINE BIOSYNTHESIS IN P. AERUGINOSA

TABLE 8. Effect on the levels of aspartic trans-carbamylase and dihydroorotase in P. aeruginosaPAO] in minimal medium in the presence of

various concentrations of 6-azauracila

Concn of 6- Gnrtotie Enzyme activitybazauracil Generation time&tg/ml) ~~~ATC'ase DHO'ase

0 55 4.2 0.910 65 4.2 0.930 81 4.6 0.840 84 4.1 0.6

a The bacteria were incubated in the presence of6-azauracil for 2 hr.

b Expressed as micromoles of product formedper hour per milligram of protein.

By analogy with similar studies on E. coli K-12(22), gross effects on the levels of aspartic trans-carbamylase would be anticipated if the first situ-ation applied. The first two enzymes, aspartictranscarbamylase and dihydroorotase were stud-ied under various conditions of growth with6-azauracil (Table 8).The generation time in minimal medium is

about 55 min, so that incubating the cells in thepresence of 6-azauracil for 2 hr allows ampletime for at least one cell division to occur. Theresults shown in Table 8 indicate that there areno concomitant increases in the levels of the bio-synthetic enzymes.

Control mechanisms other than repression.Crawford and Gunsalus (3) have shown a differ-ent type of control mechanism for tryptophanbiosynthesis in which the level of tryptophansynthetase in P. putida is controlled solely byinduction, the inducer being indoleglycerolphos-phate.One way of investigating whether such a

mechanism occurs in the uracil pathway of P.aeruginosa is to determine whether growth inthe presence of 6-azauracil results in the accu-mulation of some substrates which could theninduce one of the later enzymes; to do this, welooked at the levels of dihydroorotic acid dehy-drogenase and orotidine monophosphate pyro-phosphorylase. The results are shown in Table 9,which includes data pooled from five separateexperiments. Statistical analysis (t test) showsthat for dihydroorotic acid dehydrogenase thelevel of significance is 2%, and for orotidinemonophosphate pyrophosphorylase it is 0.1%,suggesting that the 40% rise in enzyme activityis a real effect and that partial induction of theseenzymes occurs. No such effect was found foraspartic transcarbamylase or dihydroorotase(Table 8). It thus seems possible that 6-azauracil

TABLE 9. Levels of dihydroorotic acid dehydrogen-ase and orotidine monophosphate pyrophos-phorylase after overnight growth of PAOJin minimal medium containing 6-azauracil

Concn of 6- Activity-aazauracil(sg/ml) DHOdeh'ase OMPpp'ase

0 0.24 + 0.07 0.59 4 0.0320 0.33 i 0.02 0.82 mt 0.02

Expressed as micromoles of product formedper hour per milligram of protein.

causes the accumulation, prior to dihydrooroticacid dehydrogenase, of a substrate which willslightly induce dihydroorotic acid dehydrogenaseand orotidine monophosphatepyrophosphorylase.Pinsky and Krooth (16) have reported inductionof orotidine monophosphate pyrophosphorylaseand dihydroorotic acid decarboxylase by dihy-droorotic acid in human cells (a homozygousmutant).An attempt to identify the reason for the slight

induction observed was made by growing wild-type cells in the presence of L-dihydroorotic acid(10-3M). This had no effect on enzyme levels,but it is doubtful whether the cell is permeableto dihydroorotic acid, as no mutants respond toit. Another attempt was made by examining theenzyme levels of a mutant blocked in the dihy-droorotic acid dehydrogenase step. This mutantwould be expected to accumulate dihydrooroticacid, which may then induce orotidine mono-phosphate pyrophosphorylase. The activities oforotidine monophosphate pyrophosphorylase ofmutant log phase cells in limiting and in excessuracil were 0.56 and 0.59 ,umoles per hr per mgof protein, respectively, which are not signifi-cantly different from the wild-type activity(micromoles per hour per milligram of protein)of 0.59 0.03.Feedback inhibition. Gerhart and Pardee (7)

have shown feedback inhibitions and stimula-tions of aspartic transcarbamylase from E. coliby various nucleotides and nucleosides at con-centrations of about 0.1 mm. These effects arecompetitive with respect to L-aspartate.Neumann and Jones (14) have shown aspartic

transcarbamylase from P. fluorescens to beinhibited as much as 90% by 7 mm uridine tri-phosphate and 60% by 7 mm adenosine triphos-phate, as well as lower inhibitions by othernucleotides. These inhibitions are competitivewith respect to carbamyl phosphate and non-competitive with respect to L-aspartic acid. ThepH optimum for this enzyme is 8.5, whereas the

1739VOL. 96, 1968

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ISAAC AND HOLLOWAY

pH optimum for aspartic transcarbamylase fromE. coli is 7.0.The aspartic transcarbamylase from P. aeru-

ginosa would be more likely to be similar to thatfrom P. fluorescens than from E. coli, and it wasfound that the pH optimum occurred quite closeto that for P. fluorescens, namely, pH 8.6 to 8.8.

Inhibition of the aspartic transcarbamylasefrom P. aeruginosa by various nucleotides wasexamined. Concentrations of inhibitor up to 10mM were investigated. Since it was consideredthat concentrations of 4 mm and below weremore likely to represent a physiological effect,these lower concentrations were more thor-oughly investigated.The reaction rate was determined for various

concentrations of L-aspartic acid at saturatingcarbamyl phosphate concentration (20 mM),both with and without inhibitor, and similarlyfor various concentrations of carbamyl phos-phate at saturating L-aspartic acid concentra-tions (20 mM).The inhibitions observed were found to be

competitive with respect to L-aspartate; for allsubsequent inhibition studies, the incubationmixture contained a final concentration of 20mM carbamyl phosphate and a range of L-aspar-tic acid concentrations from 0.5 mm to 20 mM.The nucleotide solutions were freshly prepared

just before use, and their stability was checkedby chromatographing a test sample of the incu-bation mixture after 30 min at 28 C and pH 8.8.No hydrolysis products could be detected, and itwas assumed that any inhibitions observed weredue to the nucleotide and not to breakdown

TABLE 10. Inhibition of aspartic transcarbamylasein P. aeruginosa PAOI by various nucleotidesa

Nucleotide addedb Per cent Rdinhibition' in

None None 1.3 mM1 mM ATP 20 2.0 mM2 mM ATP 40 2.5 mM4 mM ATP 45 3.0 mM10 mM ATP 60 5.0 mM1 mM CTP 5 1.6 mM2 mM CTP 15 1.9 mM4 mM UTP 50 3.6 mM4 mM GTP 20 2.0 mM

aConcentration of carbamyl phosphate in allincubations was 20 mm.

b ATP, adenosine triphosphate; CTP, cytidinetriphosphate; UTP, uridine triphosphate; GTP,guanosine triphosphate.

c At 0.5 mM L-aspartate.d Km calculated from Lineweaver-Burke plot.

Crude extracts were used.

products. It was also shown that the nucleotidesdo not affect the development of color involvedin assaying the product of the reaction.There was some inhibition of aspartic trans-

carbamylase by the nucleotides considered(Table 10), but it is not possible to extrapolatethe significance of these in vitro results to the invivo situation.

DIsCUsSION

This study of regulation of enzyme biosynthe-sis in Pseudomonas was commenced because ofthe nature of gene distribution and the extensivemetabolic activity of this organism. The pyrimi-dine pathway was selected because, not only hadrepression been demonstrated for both E. coliand S. marcescens, but furthermore, in E. colithe distribution of genes was such that co-ordinate repression was shown by both linkedand unlinked genes. We investigated the pattern ofrepression in P. aeruginosa, because noncluster-ing of related biosynthetic genes in this organismis more common than in E. coli.Of the limited number of biosynthetic path-

ways studied in P. aeruginosa, it appears thatrepression of biosynthetic enzymes does notoccur as generally as in certain other bacteria,nor is the magnitude of change in enzyme levelsas great when such a change is effected. Waltho(Ph.D. Thesis, Univ. of Melbourne, 1968) wasunable to demonstrate repression or derepressionfor enzymes of the phenylalanine biosyntheticpathway. However, Doy (5) showed repressionof phosphoribosyl transferase, one of the en-zymes involved in tryptophan synthesis, anotheraromatic compound. We have recently demon-strated an approximately 10-fold repression ofornithine transcarbamylase (Isaac and Hollo-way, unpublished data). In another species, P.putida, Crawford and Gunsalus (3) have studiedenzymes of the tryptophan biosynthetic pathwayand, depending on the particular enzyme, varia-tions in tryptophan availability resulted inrepression, induction, or constitutivity.Our results show, first, that the pathway of

pyrimidine biosynthesis in P. aeruginosa is thesame as in E. coli and S. marcescens, and thatthe genes concerned are not closely linked toeach other. We have been unable to demonstrateany significant changes in the levels of pyrimi-dine biosynthetic enzymes in P. aeruginosa byaltering the concentration of uracil in the growthmedium or by growth in a general rich medium.It seems, then, that these enzymes are constitutive.Furthermore, efforts to isolate mutants whichshow derepression or repression have been un-successful.

1740 J. BACTERIOL.

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

PYRIMIDINE BIOSYNTHESIS IN P. AERUGINOSA

The rate of synthesis of a particular substanceis not dependent solely on the amount of enzymepresent, as other factors, such as the presence ofinhibitors and the degree of saturation of theenzyme by its substrate, are also important.

In view of the constitutivity of the pyrimidinebiosynthetic enzymes, it would be expected thatthese other factors have considerable importancein determining the rate of synthesis of pyrimi-dines in P. aeruginosa. The substrates for aspar-tic transcarbamylase, the first enzyme unique tothe pathway, namely L-aspartic acid and carbamylphosphate, are both involved in other reactionsin the cell. If they have a relatively low affinityfor aspartic transcarbamylase, then the sub-strates are preferentially used in other biosyn-theses, unless their concentration is sufficientlyhigh to allow significant pyrimidine synthesis.

It is well known that many strains of Pseudo-monas are able to adapt to growth on a largevariety of carbon sources, and it seems that theability to induce degradative enzymes to utilizethe carbon source available is a general propertyof pseudomonads.

It is possible that this wide degradative abilitymay itself be related to the control of biosyntheticenzymes. It is plausible that, by acquiring theability to use a variety of carbon and nitrogensources, there may have been selection for genearrangements and control mechanisms which areconsiderably different from that of the clusteredoperon arrangement common amongst entericorganisms.

ACKNOWLEDGMENTS

This investigation was supported by a grant fromthe Australian Research Grants Committee.The technical assistance of Helen Barton is grate-

fully acknowledged.

LITERATuRm CITED

1. Beckwith, J. R., A. B. Pardee, R. Austrian, andF. Jacob. 1962. Co-ordination of the synthesisof the enzymes in the pyrimidine pathway ofE. coli. J. Mol. Biol. 5:618-634.

2. Chakrabarty, A. M., C. F. Gunsalus, and I. C.Gunsalus. 1968. Transduction and the cluster-ing of genes in fluorescent Pseudomonads. Proc.Natl. Acad. Sci. U.S. 60:168-175.

3. Crawford, I. P., and I. C. Gunsalus. 1966. In-ducibility of tryptophan synthetase in Pseu-domonas putida. Proc. Natl. Acad. Sci. U.S.56:717-724.

4. Demerec, M., E. A. Adelberg, A. J. Clark, andP. E. Hartman. 1966. A proposal for a uniformnomenclature in bacterial genetics. Genetics54:61-76.

5. Doy, C. H. 1964. The biochemical differencebetween certain phenotypically similar, butgenotypically different tryptophan auxotrophs

of Pseudomonas aeruginosa. Biochim. Biophys.Acta 90:180-183.

6. Fargie, B., and B. W. Holloway. 1965. Absenceof clustering of functionally related genes inPseudomonas aeruginosa. Genet. Res. 6:284-299.

7. Gerhart, J. C., and A. B. Pardee. 1962. Theenzymology of control by feedback inhibition.J. Biol. Chem. 237:891-896.

8. Handschumacher, R. E., and C. A. Pastemak.1958. Inhibition of orotidylic acid decarboxyl-ase, a primary site of carcinostasis by 6-azaura-cil. Biochim. Biophys. Acta 30:451-452.

9. Hayward, W. S., and W. L. Belser. 1965. Regu-lation of pyrimidine biosynthesis in Serratiamarcescens. Proc. Natl. Acad. Sci. U.S. 53:1483-1489.

10. Holloway, B. W. 1955. Genetic recombination inPseudomonas aeruginosa. J. Gen. Microbiol.13:572-580.

11. Holloway, B. W., and B. Rolfe. 1964. Hostgenome control in host induced modificationof Pseudomonas aeruginosa. Virology 23:595-602.

12. Lowry, D. H., N. J. Rosebrough, A. L. Farr,and R. J. Randall. 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem.193:265-275.

13. Mee, B. J., and B. T. 0. Lee. 1967. An analysis ofhistidine requiring mutants in Pseudomonasaeruginosa. Genetics 55:709-722.

14. Neumann, J., and M. E. Jones. 1964. End prod-uct inhibition of aspartate transcarbamylasein various species. Arch. Biochem. Biophys.104:438-447.

15. Pearce, L. E., and J. S. Loutit. 1965. Biochemicaland genetic grouping of isoleucine-valinemutants of Pseudomonas aeruginosa. J. Bac-teriol. 89:58-63.

16. Pinsky, L., and R. S. Krooth. 1967. Studies onthe control of pyrimidine biosynthesis inhuman diploid cell strains. Proc. Natl. Acad.Sci. U.S. 57:925-932.

17. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff.1966. The aerobic Pseudomonads: a taxonomicstudy. J. Gen. Microbiol. 43:159-271.

18. Taylor, A. L., and C. D. Trotter. 1967. Revisedlinkage map of Escherichia coli. Bacteriol. Rev.31:332-353.

19. Taylor, W. H., M. L. Taylor and D. F. Eames.1966. Two functionally different dihydro-orotic dehydrogenases in bacteria. J. Bac-teriol. 91:2251-2256.

20. Vogel, H. J., and D. M. Bonner. 1956. Acetyl-ornithinase of Escherichia coli. Partial purifi-cation and some properties. J. Biol. Chem.218:97-106.

21. Waltho, J. A., and B. W. Holloway. 1966. Sup-pression of fluorophenylalanine resistance bymutation to streptomycin resistance in Pseu-domonas aeruginosa. J. Bacteriol. 92:35-42.

22. Yates, R. A., and A. B. Pardee. 1957. Controlby uracil of fonnation of enzymes required fororotate synthesis. J. Biol. Chem. 227:677-692.

1741VOL. 96, 1968

on June 22, 2020 by guesthttp://jb.asm

.org/D

ownloaded from