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1 doi:3/nture5 Δp y Pr ric ol rb in * e c mrna fold chnge reltive to wild type in NHCl medi < / >6 Supplementl Figure. Glol nd pyrimidine-relted gene expression in ΔpyrI crb* strin.. Fold chnges of RNA trnscripts hierrchiclly clustered for ΔpyrI crb* reltive to wild type (left) nd comprtor dt of proline s nitrogen source reltive to NHCl for wild type (right). In totl, 99 genes exhiited more thn -fold chnge in the ΔpyrI crb* strin versus 6 with proline s nitrogen source suggesting comprtively minor trnscriptionl chnges result from dysregulted feedck during stedy growth.. Expression of genes in metolic pthwys relevnt to de novo pyrimidine iosynthesis. Genes re listed in the order tht the enzymes pper in the pthwy, except in the cse of urcil slvge. The rutr, purr, nd rgr genes encode repressors of their respective pthwys. c. Expression of genes most significntly ( 3 SD from men; n=) downregulted nd upregulted in the ΔpyrI crb* strin reltive to wild type. A gry r (>7 SD elow men) is shown for pyri ecuse the ΔpyrI crb* strin lcks pyri, the regultory suunit of sprtte trnscrmoylse (PyrBI), which cn ctivte or inhiit ctlytic ctivity of PyrB depending on conditions. Incresed pyrb expression my compenste for lck of PyrI ctivtion or stiliztion during stedy stte growth. Selected genes re colored to indicte their function in pyrimidine iosynthesis (pink), rginine iosynthesis (ornge), nd rut urcil degrdtion (lue). W W W. N A T U R E. C O M / N A T U R E

2 RESEARCH 6 / / /.35.. Orotte. Orotte 6 / / / 6 / / / 6 / / / 6 / / / 6 / / / 6 / / / 6 / / / UDP 6 / / / 6 / / / 6 / / / 6 / / / 6 / / / 6 / / / 6 / / / 6 / / / Dihydroorotte ATP N-crmoyl-L-sprtte Frction leled / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / Minutes post-urcil ddition Minutes post-urcil ddition Minutes post-orotte ddition Minutes post-orotte ddition Wild-type pyri::kn crb S9F pyri::kn crb S9F.35 5 N-. 5 N-. 5 N- Orotte. 5 N-Orotte Frction leled... Minutes post-urcil ddition... Minutes post-urcil ddition... Minutes post-orotte ddition... Minutes post-orotte ddition ΔpyrI crb* ΔpyrI crb* Supplementl Figure. The ΔpyrI crb * strin is defective in feedck ut not in end product homeostsis.. Metolite fold chnges following ddition of urcil (.35 or. ) or orotte (. or. ). Fold chnges were computed reltive to unsupplemented controls. The ΔpyrI crb * strin does not exhiit the chrcteristic drop in N-crmoyl-sprtte or dihydroorotte from feedck inhiition mechnisms. nd levels re nevertheless not consistently incresed. Error rs indicte stndrd devition (n=-3).. (upper) nd (lower) leling following ddition of fully 5 N-leled urcil or fully 5 N-leled orotte. The ΔpyrI crb * strin incorportes higher frction of endogenous (unleled) metolites into end products, suggesting excessive de novo flux reltive to the wild type. Error rs mrk stndrd devition (n ).

3 RESEARCH. 5 min post 5 N-orotte ddition. Leled Frction. NCM37 ΔpyrI crb* ΔpyrI crb* Δudp ΔcodA Endogenous From 5 N-Orotte Supplementl Figure 3. Leling of urcil mimics tht of uridine nd. Endogenous ( N, lue) nd orotte-derived ( 5 N, red) frctions of urcil, uridine,,, nd fter 5 minutes of 5 N-orotte supplementtion (. ). The leling pttern of urcil closely resemles tht of in the wild type. In ΔpyrI crb * strins, extrcellulr urcil nd uridine ccumulte in the stedy stte efore orotte ddition, contriuting to incresed unleled frctions of these metolites. Error rs show stndrd error (n=3-). 3

4 RESEARCH c Intrcellulr Concentrtion Intrcellulr Concentrtion Minutes post-orotte Intrcellulr concentrtion Intrcellulr Concentrtion Minutes post-orotte 7.5 min post-orotte Intrcellulr concentrtion min post-orotte ΔumpH ΔumpG ΔumpHΔumpG Δudk ΔpyrI crb* ΔudpΔcodA ΔpyrI crb*δudpδcoda ΔpyrI crb* ΔpyrI crb*δumph ΔpyrI crb*δumphδumpg ΔpyrI crb*δudpδcoda Supplementl Figure. Dynmics of overflow pthwy metolites nd pyrimidine end products following orotte ddition.. nd uridine levels following. orotte ddition. is produced over time from degrdtion of following orotte ddition. is degrded to uridine y UmpH, nd uridine to urcil y Udp. Error rs show stndrd devition (n=3).. Less urcil nd more ccumultes 7.5 minutes fter. orotte ddition in oth ΔumpHΔpyrI crb * nd ΔumpHΔumpGΔpyrI crb * strins. Error rs show stndrd devition (n=3). c. Following orotte ddition, levels re not pertured y deletion of either ΔumpH or ΔumpG individully, ut doule deletion leds to incresed. Error rs show stndrd devition (n=3).

5 RESEARCH De novo to urcil neroic net cost: 5 ATP + NADPH De novo to urcil eroic net cost: 3 ATP + NADPH Orotte to urcil net cost: ATP 3 ADP 3 ATP, NH 3 NADP + NADPH, oxlocette ATP ADP,Pi ADP ATP, RP OxPhos glutmine, ATP, HO glutmte, ADP, Pi Bicronte Crmoyl phosphte sprtte Pi N-Crmoyl-L-sprtte Dihydroorotte Q QH PRPP PPi Orotte Orotidine 5'-phosphte Pi RP Pi Crmoyl phosphte synthetse (crab) Asprtte trnscrmoylse (pyrib) Dihydroorotse (pyrc) Dihydroorotte oxidse (pyrd) Orotte phosphoriosyltrnsferse (pyre) Orotidylte decroxylse (pyrf) Phosphtse (umph/umpg) Phosphorylse (udp) Supplementl Figure 5. Energetic costs of eroic nd neroic iosynthesis nd ctolism to urcil. Relevnt rectnts nd products for indicted rections (nd corresponding gene products) re shown. Assumptions: crmoyl phosphte synthesis requires 3 ATP totl ( used y CrAB nd to regenerte glutmine from glutmte); crmoyl sprtte synthesis dditionlly requires NADPH to generte sprtte from oxlcette; susequent production of orotte produces uiquinol (QH ) whose eroic oxidtion regenertes ATP y oxidtive phosphoryltion (OxPhos); OMP synthesis requires ATP equivlents to regenerte phosphoriosyl-pyrophosphte (PRPP) from riose phosphte (RP). Shded oxes indicte de novo iosynthesis nd degrdtion to urcil (ornge), orotte conversion to urcil (lue), nd eroiclly coupled rections (yellow). 5

6 RESEARCH OD 6. +KCl or Orotte Hours x min post- 5 N-orotte +KCl (control) pacn::pyrh WT pacn::pyrh D93A pacn::pyrh N7A+D93A +orotte (. ) pacn::pyrh WT pacn::pyrh D93A pacn::pyrh N7A+D93A Ion Counts From 5 N-Orotte Endogenous. c OD 6. pacn::pyrh pacn::pyrh D93A pacn::pyrh N7A+D93A +. 5 Hours pyrh WT pyrh N7A pyrh D93A Supplementl Figure 6. The growth defects of pyrh llostery mutnts occur in diploid with the wild type llele nd re selectively oserved following orotte ddition.. Growth of pyrh diploid (genomic pyrh/ pacn::pyrh) mutnts (induced with 5 μm IPTG throughout culture) following ddition of. KCl (lines) or. orotte (shpes).. Endogenous ( N, lue) nd orotte-derived ( 5 N, red) urcil nd pek re ion counts in the ove strins t 7.5 minutes following. orotte ddition. Error rs show stndrd error (n=3). c. Growth of wild type nd pyrh WT, N7A, nd D93A in genomic ΔpyrH ckground following ddition of. urcil. 6

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