Control of Expression of the pyr Genes in Salmonella typhimurium: Effects of Variations in Uridine and

Transcription

1 JOURNAL OF BACrERIOLOGY, Mr. 1975, p Copyright 1975 Americn Society for Microbiology Vol. 121, No. 3 Printed in U.S.A. Control of Expression of the pyr Genes in Slmonell typhimurium: Effects of Vritions in Uridine nd Cytidine Nucleotide Pools MARIANNE SCHWARTZ AND JAN NEUHARD* Enzyme Division, University Institute of Biologicl Chemistry B, DK-137 Copenhgen K, Denmrk Received for publiction 24 September 1974 The differentil rte of synthesis of five of the pyrimidine biosynthetic enzymes coded for by pyrb-f, nd the endogenous concentrtions of the individul pyrimidine nucleotides were determined in specilly constructed mutnts of Slmonell typhimurium. In the mutnts employed the different pyrimidine nucleotide pools my be mnipulted individully during exponentil growth. The results obtined indicte the following. (i) The expression of pyrb, pyre, nd pyrf is controlled by uridine nucleotide in noncoordinte mnner. (ii) The expression of pyrc nd pyrd is regulted predominntly by cytidine nucleotide. Under ll conditions investigted, their expression seems to be coordinted, even though the genes re not contiguous on the chromosome. (iii) The low-moleculrweight effectors involved in controlling the expression of the pyr genes re neither uridine 5'-monophosphte nor cytidine 5'-monophosphte, but rther the corresponding di- or triphosphtes. The de novo synthesis of the pyrimidine nucleotide 5'-uridylic cid (UMP) in Slmonell typhimurium nd Escherichi coli is ctlyzed by the sequentil ction of the six enzymes: crbmylphosphte synthetse (EC ), sprtte trnscrbmylse (ATCse; EC ), dihydroorotse (DHOse; EC ), dihydroorotte dehydrogense (DHOdehse; EC ), orotte phosphoribosyltrnsferse (OMPppse; EC ), nd orotidylic cid decrboxylse (OMPdecse; EC ) (Fig. 1). The structurl genes pyra through pyrf encoding these enzymes hve been locted on the chromosomes of both S. typhimurium nd E. coli (2, 23). No close linkge hs been found between ny of the genes (Fig. 2). Regultion of the first nd the second enzyme in the pthwy, through feedbck inhibition nd ctivtion, hs been studied in gret detil (1, 2, 9, 1), wheres regultion of the pthwy t the level of gene expression hs been the subject of only few investigtions. Ytes nd Prdee (25) observed tht urcil strvtion of pyrimidine uxotrophic mutnts of E. coli led to n increse in the rte of synthesis of ll six enzymes of the pthwy. Beckwith et l. (6) determined the specific ctivities of the six enzymes in E. coli strved for pyrimidines. They concluded tht: (i) the rte of synthesis of crbmylphosphte synthetse nd ATCse ws regulted independently of ech other nd independently of the rest of the enzymes in the pthwy; (ii) the rte of synthesis of the lst four enzymes, coded for by pyrc, pyrd, pyre, nd pyrf, ws coordintely controlled. However, our present knowledge of the loction of the pyr genes on the chromosome (Fig. 2) shows tht none of them re so closely linked tht they cn constitute n operon. In previous studies (6, 24, 25), pyrimidine nucleotide limittion ws ccomplished either by urcil strvtion of pyrimidine uxotrophic mutnts or by the use of the pyrimidine nlogue 6-zurcil, which cts by inhibiting OMPdecse (21). Since, however, strvtion for UMP leds to strvtion for ll pyrimidine nucleotides of the cell (Fig. 1), most of these erlier studies did not indicte the nture of the pyrimidine nucleotide effector(s) involved in the regultion of the rte of synthesis of the individul de novo enzymes. With the isoltion of mutnts of S. typhimurium hving specific cytidine requirement (16), due to muttions in the structurl genes for cytidine deminse (cdd; EC ) nd CTP synthetse (pyrg; EC ), it becme possible to lter the cytidine nucleotide pools of the cells without, t the sme time, interfering with the uridine nucleotide pools (Fig. 1). 814

2 VOL. 121, 1975 PYRIMIDINE REGULATION IN S. TYPHIMURIUM 815 CTP - p,vr G UTP ATP+ C2+ glutmine pyra crbmyl phosphte CDP t CMP sprtote pyr8 Cytidine -- Uridine upp pyre cr bomyl sprtte Uci$ / pyrc dihydrooroe orotte sdihydroorotte p,yr D FIG. 1. Pthwy for the biosynthesis of pyrimidine nucleoside triphosphtes in S. typhimurium. The enzymes re identified by the corresponding gene designtions s follows: cdd, cytidine deminse (EC ); pyra, crbmylphosphte synthetse (EC ); pyrb, ATCse (EC ); pyrc, DHOse (EC ); pyrd, DHOdehse (EC ); pyre, OMP pyrophosphorylse (EC ); pyrf, OMPdecse (EC ); pyrg, CTP synthetse (EC ); pyrh, UMP kinse (EC 2.7.4); udk, uridine kinse (EC ); udp, uridine phosphorylse (EC ); nd upp, UMP pyrophosporylse (EC ). CDP nd UDP, Cytidine nd uridine 5'-diphosphte, respectively. pyre pyr B pyr A \s\cdd LiLI^ pyrg FIG. 2. Linkge mp of the S. typhimurium chromosome showing the loction of genes mentioned in the text. Gene designtions re explined in the legend to Fig. 1. put, Proline utiliztion (2). L d udk ' ~~~~~~~~~~~~,dpj UDP Using such double mutnts (cdd, pyrg), Abd-El-Al nd Ingrhm (1) showed tht to repress CPSse synthesis mximlly in S. typhimurium both cytidine nd rginine hve to be present in the growth medium. Thus, in ddition to rginine, cytidine nucleotide is involved in regulting the expression of the pyra gene. In the present pper we ttempt to identify the nture of the repressing metbolite(s) involved in control of the rte of synthesis of ech of the five other enzymes of the pyrimidine pthwy, i.e., those coded for by the genes pyrb through pyrf. To do this we constructed series of mutnts of S. typhimurium tht my be grown exponentilly under conditions where the composition of their pyrimidine nucleotide pools vries significntly from tht of wild-type cells. We compred the specific ctivity of ech of the five pyrimidine enzymes in the mutnts under such conditions with the pool sizes of the individul pyrimidine nucleotides. MATERIALS AND METHODS Bcteril strins. All strins employed re derivtives of S. typhimurium LT2 nd re listed in Tble 1. Growth conditions. Bcteri were grown in tris- (hydroxymethyl)minomethne (Tris)-miniml medium (8) supplemented with.15% Norite-treted vitmin-free csein hydrolyste (Csmino Acids). Glucose (.2%) ws used s crbon source. Supplementry nutrients were dded in the following finl concentrtions: L-cysteine nd L-rginine, 5 ug/ml; cytidine, 8 gg/ml; nd urcil, 5 gg/ml. Liquid cultures were grown with ertion on rotry shker t 37 C. Growth ws monitored t 436 nm in n Eppendorf photometer, model 111M. Preprtion of bcteril extrcts. Bcteri were grown exponentilly for t lest eight genertions t 37 C to n opticl density t 436 nm of.5, filtered on membrne filter (Millipore Corp.), nd wshed with

3 816 SCHWARTZ AND NEUHARD J. BACTERIOL. TABLE 1. Strins used Strin Genotype Nutritionl requirements Source or reference KP1217 cyscd519, cdd-7 Cysteine 12 JL1219 cyscd519, cdd-7, upp-22, Cysteine 12 pyrh162 JL1269 cdd-7, pyrhl69 12 JL1263 pyrh Former HD-58 (18) KP13 cyscd519, cdd-7, upp-22, Cysteine 5 cmk-15 KP1334 pyra81, pyrg16j1,c cdd-7, Arginine, urcil, nd cytidine Present work udp-2 (bove 3 C) This muttion ws formerly (18) designted pyrr. However, it hs recently been shown to reside in the pyrh gene (J. Justesen nd J. Neuhrd, unpublished dt). bnew gene designtion for the structurl gene encoding CMP kinse (5). " This pyrg muttion confers het lbility on the enzyme CTP synthetse. miniml medium. Cells were suspended in cold (4 C) miniml medium nd centrifuged. The cell pellets were kept t - 2 C overnight. After thwing, the cells were suspended in.1 M Trishydrochloride (ph 7.6)-2 mm ethylenediminetetrcetic cid to n opticl density t 436 nm of 1 to 2. The suspensions were soniclly treted for 1 min t C in n MSE ultrsonic disintegrtor. Extrcts were used for enzyme ssys without further tretment. Construction of KPl334. S. typhimurium JL155 (pyra81 pyrg166 cdd-7 udp-2), which is cytidine requiring t ll tempertures due to the pyrg166 muttion, ws trnsduced to grow in the bsence of cytidine t 3 C by using trnsducing phge P22 grown on strin contining the pyrg1611 llele. The pyrg1611 muttion confers het lbility to cytidine 5'-triphosphte (CTP) synthetse. KP1334 is still cytidine requiring t 42 C. Enzyme ssys. All ssys were performed t 37 C. Specific ctivities re expressed s units per milligrm of protein. One unit of enzyme ctivity equls 1 nmol of substrte utilized, or product formed, per minute. ATCse. ATCse ws ssyed by the method of Gerhrt nd Prdee (1), except tht the ssys were performed t 37 C. DHOse, DHOdehse, OMPppse, nd OMPdecse were ssyed by the method of Beckwith et l. (6) with the modifictions described below. DHOse. For DHOse, the ssy mixture contined in.5 ml:.1 M Tris-hydrochloride (ph 8.8),.2 mm ethylenediminetetrcetic cid, 2 mm L- dihydroorotte, nd bcteril extrct. The mixture ws kept in ice bth for 5 min nd then trnsferred to 37 C. At 2, 12, nd 22 min, 15-ul smples were removed, nd the mount of crbmylsprtte formed ws determined s in the ATCse ssy. DHOdehse. For DHOdehse, the ssy mixture contined in 1 ml:.1 M Tris-hydrochloride (ph 8.8), 6 mm MgCl2, nd bcteril extrct. L-Dihydroorotte (5 Il, 2 mm) ws dded to strt the rection. The conversion of L-dihydroorotte to orotte ws followed continuously t 29 nm in Zeiss-PMQ III spectrophotometer. An increse in bsorbncy of 1.93 (1-cm light pth) is equivlent to chnge in substrte concentrtion of 1 mm. OMPppse. For OMPppse, the ssy mixture contined in 1 ml:.1 M Tris-hydrochloride (ph 8.8), 6 mm MgCl2,.25 mm orotte, nd bcteril extrct. The rection ws strted by ddition of 1 Al of 6 mm 5-phosphoribosyl-1-pyrophosphte. The rection ws followed spectrophotometriclly t 295 nm. A decrese in bsorbncy of 3.67 is equivlent to n increse of OMP concentrtion of 1 mm. OMPdecse. The ssy mixture contined in 1 ml:.1 M Tris-hydrochloride (ph 8.8), 6 mm MgCl2, nd bcteril extrct. OMP (1 ul,.2 M) ws dded to strt the rection. The rection ws followed t 29 nm. A decrese in bsorbncy of 1.38 is equl to decrese in OMP concentrtion of 1 mm. Proteins. Proteins were determined by the method of Lowry et l. (15), with bovine serum lbumin s stndrd. The crude extrcts used for the enzyme ssys were spun for 5 min t 6, x g. The superntnt ws used for protein determintions. Nucleoside triphosphte pools. The nucleoside triphosphte pools were determined in cultures grown for two genertions in the presence of [92P Jorthophosphte (specific ctivity, 1 gci/gmol). Growth conditions in the lbeled cultures were exctly the sme s in the cultures used for enzyme determintions. Extrction nd chromtogrphic techniques used for determining the individul nucleoside triphosphte pools hve been described previously (17). To void vrition in the clculted pool sizes due to smll chnges in the concentrtion of orthophosphte in the medium, ll pools re given in percent totl ribonucleoside triphosphtes. RESULTS The vilbility of S. typhimurium mutnts blocked in different steps of pyrimidine nucleotide interconversion (12, 19) hs mde possible the construction of strins in which the different pyrimidine nucleotide pools my be mnipulted independently. A study of the effects of such chnges in individul pyrimidine nucleotide pools on the levels of the pyrimidine

4 VOL. 121, 1975 PYRIMIDINE REGULATION IN S. TYPHIMURIUM 817 biosynthetic enzymes should help in identifying the prticulr pyrimidine nucleotide effector(s) involved in regulting the rte of synthesis of ech of the enzymes coded for by the genes pyrb through pyrf. The mutnts used in the present study disply the following unique chrcteristics. (i) They re cpble of growing exponentilly under conditions in which their individul pyrimidine nucleotide pools vry from tht observed during growth of wild-type cells. (ii) They do not contin muttions in ny of the genes pyrb through pyrf encoding the five enzymes under investigtion. Effect of vrition in the uridine nucleotide pools. Muttions in the structurl gene for UMP kinse (pyrfl); rendering this enzyme prtilly defective result in cells tht will grow with reduced uridine 5'-triphosphte (UTP) nd uridine 5'-diphosphte pools. The size of the UTP pool, s well s the growth rte of ny prticulr mutnt, is dependent on the pyrh muttion in question (12). In Tble 2, experiments 1, 4, 5, nd 6 give the nucleotide triphosphte pools nd the growth rtes of pyrh+ strin JL1217 nd of three different pyrh mutnts JL1263, JL1219, JL1269, respectively. From this tble it ppers: (i) tht the growth rte of the three pyrh mutnts decreses with decrese in the reltive UTP pools, (ii) the reltive CTP pools do not chnge in prllel with the UTP pools, nd (iii) ddition of exogenous urcil to the pyrh+ strin results in definite increse in the UTP pool of the cells (experiment 2). A similr increse is not observed in pyrh mutnts due to their defective UMP kinse (not shown). Tble 3 lists the specific ctivities of the five pyrimidine biosynthetic enzymes in strin JL1217 grown in the presence nd bsence of urcil, nd in the three pyrh mutnts. There is n inverse reltionship between the reltive ctivity of ech of the five enzymes nd the size of the UTP pool, suggesting tht uridine nucleotide my be involved s n effector in the regultion of the rte of synthesis of these enzymes. For three of the enzymes this is illustrted grphiclly in Fig. 3. The results from Tble 3 show lso tht the rte of synthesis of individul enzymes derepresses to vrious degrees. Mximl vlues obtined (rtio of highest to lowest) re 19-fold for ATCse, 14-fold for DHOse nd DHOdehse, 32-fold for OMPppse, nd 7-fold for OMPdecse. Effect of vritions in the cytidine nucleotide pools. Cytidine dded to the medium of wild-type S. typhimurium or E. coli is quntittively converted to uridine by the cells. The demintion is ctlyzed by the periplsmic enzyme cytidine (deoxycytidine) deminse (cdd) (3, 16, 19; Fig. 1). In contrst, cdd mutnts convert exogenous cytidine to CTP effectively. All of the mutnts employed in the present study contin the muttion cdd-7 (Tble 1). The effect of exogenous cytidine on the reltive nucleoside triphosphte pools of strin JL1217 is shown in Tble 2 (experiments 1 nd 3). Cytidine cuses significnt increse in the CTP pool without ffecting the UTP pool. The effect is even more striking in strin JL1269, in which the UTP pool is very low due to the pyrh muttion (Tble 2, experiments 6 nd 7). In this strin, the ddition of cytidine results in CTP pool greter thn the denosine 5'-triphosphte TABLE 2. Growth rtes nd reltive pool sizes in different mutnts of S. typhimurium Addition to Reltive pool sizes, Expt Strin genotype lent growth medium (4g/ml) Gowth doubling/h UTP CTP ATP GTP 1 JL1217 None JL1217 Urcil, JL1217 Cytidine, JL1263 pyrh None JL1219 pyrh None JL1269 pyrh None JL1269 pyrh Cytidine, KP13 cmk None KP1334 pyrg None The relevnt genotype nd pyrimidine ddition for ech of the experiments re given. For further informtion see Tble 1. bpercent totl ribonucleoside triphosphte pool (NTP,O,). In the experiments shown, NTP,O, ws (in micromoles per grm of bcteril dry weight): JL1217, 14; JL1263, 1; JL1219, 9; JL1269, 35; KP13, 9; KP1334, 29. ATP, Adenosine 5'-triphosphte; GTP, gunosine 5'-triphosphte.

5 818 SCHWARTZ AND NEUHARD J. BACTERIOL. TABLE 3. Levels of the five pyrimidine biosynthetic enzymes during UTP limittion Addition to Pool sizes Reltive sp ctb Expt. T Strin medium UTPoCTP CTP ATCse DHOse DHOdehse OMPppse OMPdecse (6g/ml) pyrb pyrc pyrd pyre pyrf 1 JL1217 Urcil, JL1217 None (48) 1 (5) 1 (5) 1 (55) 1 (45) 3 JL1263 None JL1219 None JL1269 None Pool sizes re tken from Tble 2. b Specific ctivities re expressed reltive to the specific ctivity found in JL1217 (experiment 2). Vlues in prenthesis re the bsolute specific ctivities expressed s nnomoles per minute x milligrm of protein. For ech strin the sme extrct ws used for ll the enzyme ssys. The figures given re the verge of two determintions on the sme extrct. I- 11 LL w c-j CY w 4x 6 7 s 5 4 3F 2 o ClI1% 1i, /o CTP o-o ATCse A OMPppse 4 13 * 1 A * o o \1 I I I i ~ ec - so OMP decse / UTP *-u FIG. 3. Derepression of sprtte crbmyltrnsferse (ATCse), OMPppse, nd OMPdecse in mutnts of S. typhimurium growing exponentilly with ltered UTP nd/or CTP pools. The ordinte represents specific ctivities reltive to those found in strin JL1217 growing in bsence of exogenous pyrimidines. The bsciss represents the UTP pool (bottom) or CTP pool (top) in percent totl ribonucleoside triphosphte pool. Symbols: *, reltive specific ctivities plotted versus the UTP pools;, reltive specific ctivities plotted versus the CTP pool. The dt re tken from Tbles 3 nd 4. o 1- pool without significntly ffecting the reltive size of UTP. Tble 4 (experiments 1 through 4) gives the specific ctivities of the five enzymes in strins JL1217 (pyrh+) nd JL1269 (pyrh169) grown with or without cytidine dded to the medium. The dt suggest tht high CTP pool is ccompnied by significnt repression of only two of the enzymes of the pthwy (i.e., DHOse nd DHOdehse) nd tht the rte of synthesis of these two enzymes is repressed by the ddition of exogenous cytidine both in strins growing with norml UTP pool (JL1217) nd in strins growing with very low UTP pools (JL1269). To confirm the selective effect of chnges in cytidine nucleotide pools on the rte of synthesis of DHOse nd DHOdehse, we employed

6 VOL. 121, 1975 PYRIMIDINE REGULATION IN S. TYPHIMURIUM 819 mutnt KP1334, which contins het-lbile CTP synthetse (pyrg1611) (Tble 1). At 37 C in the bsence of cytidine, the growth of KP1334 is somewht slower thn tht of pyrg+ strin. Under these conditions the reltive CTP pool of the cells is very low, while the UTP pool is incresed (Tble 2, experiment 9). From Tble 4 (experiment 5) it ppers tht low CTP pool is ccompnied by n increse in the rte of synthesis of DHOse nd DHOdehse. Nucleoside mono-, di-, or triphosphte? From the results presented in the previous sections it ppers tht vritions in the uridine nucleotide pool or the cytidine nucleotide pool hve different effects on the rte of synthesis of ech if the five pyrimidine biosynthetic enzymes. We would like to estblish the exct ech cse nture of the effector nucleotide in nd thus determine the level of phosphoryltion of the effector nucleotide(s). This requires tht we modulte the rtio between the pyrimidine nucleoside mono-, di-, nd triphosphtes in vivo nd study the effect of such modultions on the differentil rte of synthesis of the individul enzymes. In the present study, uridine nucleotide limittion is ccomplished by the use of pyrh mutnts with high levels of UMP due to their prtil defect in UMP kinse. Thus, we conclude tht the effector involved in the regultion of the pyr genes is not UMP, but rther uridine 5'-diphosphte, UTP, or eventully uridine sugr nucleotide. Recently, we isolted nd chrcterized mutnt of S. typhimurium, KP13, lcking cytidine 5'-monophosphte (CMP) kinse ctivity (5). Mutnt KP13 hs slightly lower growth rte thn strin JL1217. Tble 2 shows tht the UTP nd CTP pools of KP13 re nerly identicl to those of the wild type. TABLE 4. However, the CMP pool is sixfold higher in the mutnt thn in the prent strin (5). Since the specific ctivities of DHOse nd DHOdehse re not lower in the mutnt thn in the prent strin (Tble 4), we conclude tht the cytidine nucleotide effector involved in repressing the rte of synthesis of DHOse nd DHOdehse is not CMP but rther cytidine 5'-diphosphte or CTP. DISCUSSION No close linkge is found for ny of the pyrimidine genes (pyra through pyrf) encoding the six enzymes for the biosynthesis of UMP in S. typhimurium nd E. coli (Fig. 2). The expression of these genes is controlled by pyrimidines; pyrimidine strvtion results in derepression of the rte of synthesis of ll six enzymes. Previous results obtined with E. coli indicte tht, wheres the expression of pyra nd pyrb ws regulted independently, the expression of the four remining genes seemed to be coordintely regulted (6, 25). Recently, however, Dennis nd Hermn (7) found tht prtil pyrimidine strvtion of pyrb mutnt of E. coli, creted by feeding orotic cid s the sole pyrimidine source, resulted in noncoordinte derepression of the rte of synthesis of DHOdehse (pyrd) nd OMPppse (pyre). In ll the bove-mentioned studies, pyrimidine limittion implied strvtion of the cells for UMP, which in turn results in more or less generl strvtion for ll endogenous pyrimidine compounds. The isoltion of mutnts in S. typhimurium hving growth requirement for cytidine (pyrg, cdd) llowed the mnipultion of the cytidine nucleotide pool without interfering with the uridine nucleotide pool. Abd-El-Al nd Ingrhm (1) used such mutnts to show Levels of five pyrimidine biosynthetic enzymes with vritions in the CTP pool Addition to Pool sizes Reltive sp ct' Expt Expt Strin growth medium UTP CTP ATCse DHOse DHOdehse OMPppse OMPdecse (,Og/ml) pyrb pyrc pyrd pyre pyrf 1 JL1217 Cytidine, JL1217 None (48) 1 (5) 1 (5) 1 (55) 1 (45) 3 JL1269 None JL1269 Cytidine, KP1334 None KP13 None Pool sizes re tken from Tble 2. b Specific ctivities re expressed reltive to the specific ctivity found in JL1217 (experiment 2). Vlues in prenthesis re the bsolute specific ctivities expressed s nnomoles per minute x milligrm protein. For ech strin the sme extrct ws used for ll the enzyme ssys. The figures given re the verge of two determintions on the sme extrct.

7 82 SCHWARTZ AND NEUHARD J. BACTERIOL. tht in order to obtin full repression of pyra both rginine nd cytidine hd to be present in the growth medium. The presence of urcil did not ffect the rte of synthesis of crbmylphosphte synthetse significntly. Using similr mutnts (pyra, pyrg, cdd), Willims nd O'Donovn (24) studied the derepression of four other genes pyrb, pyrd, pyre, nd pyrf. They determined the specific ctivities of ATCse, DHOdehse, OMPppse, nd OMPdecse fter 1-h strvtion period for either cytidine or urcil. From their results it ppered tht, wheres strvtion for urcil (not for cytidine) resulted in significnt increse in the specific ctivity of ATCse, only cytidine strvtion promoted incresed levels of DHOdehse, OMPppse, nd OMPdecse. Since, however, their results were obtined with nongrowing cell popultions nd no simultneous determintions of the individul intrcellulr pyrimidine nucleotide pools in the strved cells were performed, it is difficult to mke ny firm conclusions regrding the differentil rte of synthesis of the individul enzymes investigted or the nture of the effector involved (cytidine or uridine nucleotide) in controlling the expression of the genes pyrb, pyrd, pyre, nd pyrf. In the present pper we hve likewise studied mutnts in which uridine nucleotide nd cytidine nucleotide metbolism is prtilly seprted by muttion, i.e., in cdd mutnts. However, the mutnts employed by us contin dditionl muttions tht mke it possible to grow these strins exponentilly under conditions where the individul pyrimidine nucleotide pools differ significntly from those of the prentl strins. This enbles us to compre the difterentil rte of synthesis (i.e., specific ctivity during exponentil growth) of the five enzymes encoded for by the genes pyrb through pyrf with the intrcellulr levels of the individul pyrimidine nucleotides. The results obtined (Tbles 2 through 4 nd Fig. 3) my be summrized s follows. (i) The rte of synthesis of ll five enzymes investigted is incresed in cells growing with decresed UTP pools. The extent of derepression vries inversely with the size of the UTP pool (Tble 3). However, the extent of derepression observed vries considerbly for the individul enzymes. Since the results were obtined with mutnts contining prtilly defective UMP kinses, their UMP pools re high nd, thus, UMP my be ruled out s n effector in the control of expression of the corresponding structurl genes pyrb through pyre. (ii) The differentil rte of synthesis of ATCse, OMPppse, nd OMPdecse responds qulittively in the sme wy to chnges in pyrimidine nucleotide pools. This is illustrted in Fig. 3, which shows the levels of these enzymes in exponentilly growing cells of different mutnts s functions of the CTP pools or the UTP pools of the mutnts. Moreover, the results presented in Fig. 3 suggest tht the rtes of synthesis of these three enzymes re under control of UTP or uridine nucleotide closely relted metboliclly to UTP (e.g. uridine 5'- diphosphte or uridine 5'-diphosphte sugr) rther thn under control of CTP. (iii) The rte of synthesis of DHOse nd DHOdehse seems to respond to vritions in both UTP nd CTP, since limittions for either of these nucleotides cuse derepression (Tble 3; Tble 4, line 5). However, the finding tht high CTP pools re ccompnied by full repression of the synthesis of DHOse nd DHOdehse independent of the size of the UTP pools (cf. JL1217 nd JL1269 with cytidine, Tble 4, experiments 2 nd 4) suggests tht CTP control is "dominnt" over UTP control. The results obtined with KP13, lcking CMP kinse ctivity (Tble 4, experiment 6), indicte tht the cytidine nucleotide involved in the regultion is not CMP but rther cytidine 5'-diphosphte or CTP. It should be noted (s illustrted in Fig. 4) tht, under ll conditions tested, the expression of the pyrc nd pyrd genes seems to be coordinte. Since the two genes re seprted by severl unrelted genes (e.g., the put genes [2]; Fig. 2), they do not comprise n operon. However, it is suggested tht they shre common repressor nd tht their promotoropertor regions re very similr. Ginther nd Ingrhm (11) hve recently isolted cold-sensitive mutnt of S. typhimurium, JL297, defective in the structurl gene coding for nucleoside diphosphokinse (ndk; EC ). Studies of strin JL297 (11) indicted tht cesstion of growth of the mutnt t the nonpermissive temperture (2 C) results in 5% decrese in the nucleoside triphosphte pools nd no ccumultion of nucleoside diphosphtes. Under the sme condition, the rte of synthesis of ATCse nd DHOdehse (no other pyrimidine enzymes were ssyed) ws repressed. An explntion for these unexpected results my be relted to the observtion tht t the nonpermissive temperture strin JL297 ccumulted significnt mounts of ppgpp nd pppgpp (11). Severl ttempts hve been mde to isolte mutnts showing constitutive synthesis of ll

8 VOL. 121, 1975 PYRIMIDINE REGULATION IN S. TYPHIMURIUM 821._ m, 1 u._ u._ U) do I Go Ut DHOdehse reltive specific ctivity FIG. 4. Coordinte expression of the pyrc nd pyrd genes. The reltive specific ctivities of DHOse re plotted ginst the reltive specific ctivity of DHOdehse in different mutnts of S. typhimurium growing exponentilly with ltered UTP nd CTP pools. The dt re tken from Tbles 3 nd 4. the pyrimidine biosynthetic enzymes, either by selecting for resistnce to pyrimidine nlogues or by selecting for pyrimidine overproducing mutnts (18). However, ll of the mutnts isolted hving high levels of the enzymes, when grown in the presence of exogenous urcil (designted pyrr [18]), hve recently been chrcterized s mutnts defective in the structurl gene pyrh coding for UMP kinse (J. Justesen nd J. Nevihrd, unpublished dt). Therefore, no mutnts directly ffected in the regultion of the expression of ll the pyr genes hve been chrcterized. This, together with our present results, leds us to believe tht t lest two repressors re involved in controlling the expression of the pyr genes. Evidence in two biosynthetic systems hs demonstrted tht the first enzyme in pthwy cn ply role in the repression of enzymes in tht pthwy (13, 14). Such mechnism does not seem to be opertive in the repression of the pyrimidine pthwy since it hs been shown (22; M. Schwrtz, unpublished dt) tht deletion muttion tht covers the entire pyrb locus in S. typhimurium does not ffect the repressibility or the derepressibility of the residul enzymes of the pthwy. However, this need not exclude the following possibilities: (i) tht the pyrb gene product is involved in its own expression; or (ii) tht the gene product of ny of the other pyr genes could be involved in the regultion of its own synthesis or in the regultion of the synthesis of certin of the other enzymes of the pthwy. ACKNOWLEDGMENTS We would like to express our grtitude to J. L. Ingrhm in whose lbortory this work ws initited. Some of the preliminry experiments on ATCse nd DHOdehse were done with C. F. Beck. The expert technicl ssistnce of Lise Schck is gretly cknowledged. This work ws supported by grnts from Sttens nturvidenskbelige Forskningsrd nd from NATO (no. 629). LITERATURE CITED 1. Abd-El-Al, A., nd J. L. Ingrhm Control of crbmyl phosphte synthesis in Slmonell typhimurium. J. Biol. Chem. 244: Anderson, P. M., nd A. Meister Control of Escherichi coli crbmyl phosphte synthetse by purine nd pyrimidine nucleotides. Biochemistry 5: Beck, C. F., nd J. L. Ingrhm Loction on the chromosome of Slmonell typhimurium of genes governing pyrimidine metbolism. Mol. Gen. Gent. 111: Beck, C. F., J. L. Ingrhm, J. Neuhrd, nd E. Thomssen Metbolism of pyrimidine nd pyrimidine nucleosides by Slmonell typhimurium. J. Bcteriol. 11: Beck, C. F., J. Neuhrd, E. Thomssen, J. L. Ingrhm, nd E. Kleker Slmonell typhimurium mutnts defective in cytidine monophosphte kinse (cmk). J. Bcteriol. 12: Beckwith, J. R., A. B. Prdee, R. Austrin, nd F. Jcob Coordintion of the synthesis of the enzymes in the pyrimidine pthwy of E. coli. J. Mol. Biol. 5: Dennis, P. P., nd R. K. Hermn Pyrimidine pools nd mcromoleculr composition of pyrimidinelimited Escherichi coli. J. Bcteriol. 12: Edlin, G., nd. Mloe Synthetis nd brekdown of messenger RNA without protein synthesis. J. Mol. Biol. 15: Gerhrt, J. C A discussion of the regultory properties of sprtte trnscrbmylse from Escherichi coli, p In B. L. Horecker, nd E. R. Stdtmn (ed.), Current topics in cellulr regultion, vol. 4. Acdemic Press Inc., New York. 1. Gerhrt, J. C., nd A. B. Prdee The enzymology of control by feedbck inhibition. J. Biol. Chem. 237: Ginther, C. L., nd J. L. Ingrhm Cold-sensitive mutnt of Slmonell typhimurium defective in nucleoside-diphosphtekinse. J. Bcteriol. 118: Ingrhm, J. L., nd J. Neuhrd Cold-sensitive mutnts of Slmonell typhimurium defective in uridine monophosphte kinse (pyrh). J. Biol. Chem. 247: Kovch, J. S., M. A. Berberich, P. Venetiner, nd R. F. Goldberger Repression of the Histidine operon: effect of the first enzyme on the kinetics of repression. J. Bcteriol. 97: Levinthl, M., L. S. Willims, M. Levinthl, nd H. E. Umbrger Role of threonine deminse in the regultion of isoleucine nd vline biosynthesis. Nture (London) 246: Lowry,. H., N. J. Rosebrough, A. L. Frr, nd R. J.

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