Supporting Information Vegetative Cell and Spore Proteomes of Clostridioides difficile show finite differences and reveal potential protein markers. Wishwas R. Abhyankar1, 2, Linli Zheng1, 2, Stanley Brul1*, Chris G. de Koster2†, Leo J. de Koning2 1Department of Molecular Biology and Microbial Food Safety, University of Amsterdam, Amsterdam, the Netherlands; 2Department of Mass Spectrometry of Bio-Macromolecules,University of Amsterdam, Amsterdam, the Netherlands. † Deceased August 5th, 2019. *Corresponding author E-mail: [email protected] Table of Contents Supplementary Figure S1. Cellular overview of quantified proteins from spores and vegetative cells of C. difficile 630. Supplementary Figure S2. Pathways and proteins shared by the vegetative cells and spores of C. difficile 630. Supplementary Figure S1. Cellular overview of quantified proteins from spores and vegetative cells of C. difficile 630. The overview is generated using the BioCyc pathway analysis tool. The quantified proteins that are spore-predominant (red), commonly shared yet spore-enriched, commonly shared but cell-enriched (grey) and cell-predominant proteins (purple) are represented with the pathways to which they belong. Refer to Supplementary Table S1 and Supplementary Figure S2 for more details. os o os p o o l-hist1dme b10synthes1S os o o L·homosenneand L·meth1onuie b1osynthesis superpathway of L·tyrosine b1osynthes1s o superpathwayof L-meth1ornne ATP b1osynthes1s (transsutfurat1on) l D-erythr e PAPP ph ph enol as xal acetate pyruv 4-p phate xal acetate chorismate oxal acetate osaspartate kinase I: dapG aspartate aspartate l phospho-2- aspartale bifunclionaJ I aspartokinase: ysC aminotranslerase: ammotransferase: aspC os phosphoribosyltrans1erase:o � aspC hisG dehydro-3- aminotransrerase: chorismate r os deoxyheptonate a,pC aspartate l mutasel L-aspartyl-4-ph phate aspartate aldolase:aroF aminotranslerase: aspB prephenate aspartale dehydratase: aminotranslerase: t-(5-ph ph - aminotransrerase: aspartate am!notransferase· aspB � j pheA aspartate-semiatdehyde -D- ib yl) phospho-2- asps CD630_25l50 dehydro-3· s sl dehydrogenase:asd aspartate deoxyheptonate p aspartate aminotrans!erase: ::; aldolase: aroF aspartate CD630_28280 prephenate aminotrans!erase: o aminotranslerase: !i� CD630_25t50 E oso L-a partate- emialdehyde r.� CD630_25150 phosphoribosyl·ATP., o as l bltuocllonat l 3-d xy- aspartate pyrophosphatase: aspartate chorismate homo,enne aminotranslerase: hisl D-arabin ­ aminolranslerase: mutase/ p aspartate kmase L dapG j dehydrogenase:hom1 CD630_26260 D�7iifo��1f.�'M�- heptul nate CD630_28280 l prephenate 7-ph phate s l aspartokinase:os ysC dehydratase: homoserine o o pheA o os dehydrogenase:hom2 asp 1 3·dehydroquinate l lo as p p l I synthase:aroB l-a partyl-4-ph phate a,partate I l kmase brluncbonal a,pa,tate 2- x -3- L-h m erine :dapG phosphonbosyl kinase henyl anoate j 3-dehydrl quinate l:dapG aspartate-semlaldehyde homoserine 0- j dehydrogenase:asd p aspartokinase: ��:sp�i�;���� l s o os r "-- succiny translerase: metA s lysC 3-dehydroquinate aspartokinase: j pyrophosphatase: rosh s os dehydratase:aroD lysC hIsI p p L-aspartate-semialdehyde he l-a partyl- m cy -L-h m erine 3-d <,a� + 4-phosphate J;\�;fif1-(5 ph ph @ D-nbosyl)- L-a partyl- homo,ertne � ;if�'��' s o � 4- hos hate j dehydrogenase: hom1 8spMate­ a,partate­ homoserine l semialdehyde �: o os dehydrogenase:hom2 dehydrogenase· semialdehyde L-cy tathi nine s r a,o l dehydrogenase: l s a,d ] I bilunctional cystathionine dehy���i�nase: L-h m erine s beta-lyaselmaltose l-a pa tate- methylldeneamino] regulon repressor: ma!Y sem;aldehy im!dazole-4- L-a partate- homoserine 0- :: r carboxamide sem;aldehy s "-- succinyltranslerase:o os metA mo,eooe os o os or isomeraseo : hisA hikimate :: dehydrogenase: os mo,e.,ne dehydrogenase: homl shikimate homocysteine S­ l hom1 cy -L-h m erine methyltranslerase: kmase: i j homoserine ph ph ribul ylf mimin ­ s aroK � CD630_35960 dehydrogenase: AICAA-ph phate os j homoserine s o o s r h0m2 dehydrogenase: o o hom2 hikimate 3- lmldazole ph phate L-cy tathi nine glycerol l l-h mo e ine phosphate l 3-phosphoshikimate 1- L-h m serine blfunctional cystathionine synthase: o carboxyvinyllranslerase:o l beta-Iyase1maItose homoserfne os o hIsHhisF o aroA kinase: thrB o homoserineo regu on repressor: malY o os o kinase:thrB l o os superpathway of branched chamamino acid b1osynthes1s 5-en lp ruv yl­ O-ph ph ­ -erythr - l l shikimate 3- l-h m erine AICAA O-ph sph ­ pyruvate lmid � -� 1 er l- phos hate homocysteine S­ thr � �� � � L-h m erine l l uueonloe chorismate methyltransferase: acetolactate lmidazo eglycero synthase: l threonioe CD630_35960 l ­ synthase: synthase large o threonineo o sl thrC o ophosphate a,OC synlhase: o subunit:ilvB dehydratase dehydratase: hisB thrC 11:tdcB osI (2Z)-2-amin but-2-en ate tht lmidaz le acet l- ch ri mate {S)-2-acet lactate 1 thr l ph pha1e o o olhreomne bifunct�oal l ketot-a,� o histidinol-phosphate chorismate reducto.isomerase:ro dehydratase l 11:tdcB aminotranslerase: mutase/ o ,I,c hisC l prephenate 2-imin butan ate (22)-2-amin but- dehydratase: 1 I o (2A)-2,3-dihyd xy­ 2-en ate histidino -phosphate r pheA s o !3-methylbutanl ate aminotranslerase: s hisC dihydroxy-acid dehydratase: ilvD 2-oxobutan ate p ephenate 2-iminobutanoate l-hi tidin l­ 1 pho phate 1 acetolactate 1 prephenate IMNMiH synthase large os o o o o dehydrogenase: subunit:ilvB histidinol­ o ty,C p ro o phosphatase: hiSK l 2- xobutan ate 3-ph ph - (S)-2-acet -2- l histidinol 4-hydr xyphenylpyruvate D-glycerate _ _ so o hyd xybutan ate phosphatase/ o o 0 3 1 acetola""" hydrolase: phosphog ycerate l ketol-acid synthase s o 1 l dehydrogenase: serA reductoisomerase:ro CD630_10690 arge. _ a,part:.: a,pa,agine (2S)-2-i pr pylmalate o l l o flvC subunIt:Ilv8 -ammonia synthetase: o o 3-ph sph - ligase: asnA asnB tyr hydrox rruvate 3-isopropytmalate hi tidin l so ro dehydratase: (A)-2,3-dihyd xy- l os o asparagine asparagine {S)-2-acet -2- r euc leuD 3-methylpentanl ate sy synthetase: l synthetase: hydr xybutan ate s histidinol asnB asnB dihydroxy-acid dehydrogenase: 2-i p pylmaleate o o o ketol-acld 1 l dehydratase: ilvD l hisD 3-ph ph ­ asparagine reductoisome ase: o l- erine syn!hetase: ilvC so ro3-isopropylmalate o hi tidinat asnB dehydratase: l l asn euC leuD (S)-3-methyl-2- x pentan ate (A)-2,3-dihydr xy- histidinol 3-methy!pentan ate s dehydrogenase: (2A,3S)-3-i lp pylmalate hisD l dihydroxy-acid asn l so 3-o isopropylmatate dehydratase: o os dehydrogenase: ilvD hi serinel l ile p teuB hydroxymethyltranslerase:g yA senne (2S)-2-i pr pyl- (S)-3-methyl-2· hydroxymethy translerase: g yA 3- x uccinate1 oxo e noate o o o r gly 4-methyl-2- x pentan ate l#;i#iffiHe 1MI I os os leu D-rib e 5- Scale representing the ph lphate Log (14N/15N) ratios nbose-phospha\e 2 pyrophosphokmase: prs PAPP superpathway of purine nucleondes de novo b1osynthests I superpathway of pynmidine nbonucleot1des de novo b10synthesis superpathway ot punne nucleotides de novo brosynthests II PRPP PAPP gin hydrogencarbonate amidophosphont>Osyltranslerase:purF am;dopMsp,o,;bOsy,oans<,,asec P"'' carbamoy1-phosphate ! l synthase· carB1 carA carbamoyl-phosphate o gly synthase: carB2 carA J:ff�t:i� ·jl· o gty phospnor,oosy1amine­ J3ctf.�g:0��- - carbamoyl ph phate asp ·glycme ligase: purD phosphon�osylamine­ ·glycme hgase pu,o aspartate � carbamoyltranslerase py1B GAR GAR phosphoribOsylglycinam!de l rormyltrans1erase:purN N-carbamoyl-L·aspartate FGAR 1 d1hydroorotase:pyre FGAA priospnorib0sy1lormy1g1ycinam1dlne dihydroorotase: synthase: purL phosphor1bosyllormylglycinamid1ne CD630_36430 1 synthase: purL l FGAM 1 (S)-dihydroorotate FGAM phosphoribosyllormylglycinamldine cyclo-ligase: purG phosphoribosyllormylglycinamidine l cyclo-ligase:purG RPP 1 o,otat AIR 1 AIR orotate A;R Y-phosphonbosyltranslerase· carbOxylase: pyrE l C0630_0l950 carboxy1ase: C0630_01950 N5·CAIR orotidine 5'·phosphate A;R asp NCA;R m"<a,ec carboxylase: C0630 01950 purE orotidine 5'-phosphate decarboxylase: pyrF 5-carn:xyamlnolmldazore l nbonucleotJde mutase:pure l CAI UMP j "" uridylate kinase: pyrH i CAIR phosphorlbosylaminoimidazolesucclnocarboxamide synlhase:purC UDP pnospnorit>OsyramJnolmidazoiesuccinocarbOxamide syntnase:purC phosphonbosylam,noimlaa:rolesucc,nocarboxam,de synlhase: C0630_04890 phosphoribosyraminoimidazotesuccinocarboxamide synthase: C0630_04890 SAICAA SAICAAl gin adenylosucc1nate UTP adenylosuccJnate lyase:purB '-- lyase: purB 1 AICAA 1 CTP AICAA o;t"="ona, """nc,;ona, phosphoribOsytamlnolmlda:roleca,bOxamide phosphoribosylamJnolmlda:roteca1boxamlde lormyltransferas@IIMP cycrohydrOLas@: purH lormyltransleras&IIMP cyclohydrolase:purH 5-aminoimidazore- 5-am,noimidazole- 4-carboxamlde 4-carboxamide nbonucleotlde nbonucleollde translormylase translormylase: CD630_08410 C0630_064 10 l l FAICAR FAICAA bilunc1ional bllunclional phosphorlbosylaminolmldazolecarboxamide phosphonbosylamlnolmlda:rorecarbOxamlde lormyltranslerase/lMP cyclohydrolase: purH lormyltranslerase/lMP cyclohydrolase: purH asp IMPl IMP asp j superpathway of adenosme adenylosucclnate nucleotides de novo b1osynthesis I Jnosine 5- inosine 5- synthetase. purA monophosphate monophosphale denyarogenase: aenyarogenase: guaB guaB adenylo-succinate adenylo-succinate adeny,os"cdna<e adeny<os"cdaa<e 1yase: pure l XMP lyase:pu1B l adenylo·succinate XMP GMP AMP adeny,os=c,na<e syntnase· AMP guaA lyase: purB GMP adeny<a<e synthas@: adenylate l 9"aA kmase:adk kmase· adk l l GMPl l AMP GMP ADP adeny<a<e kmase: gmk kinase:adk g"any<a<e l •"··,···· l kinase:gmk ribonucleoside-