, !toxin!genes!might!be!differentially!expressed!in!the!gut.! 703! Accordingly,! formate! and! acetate! (directly! obtained! from! pyruvate)! predominate! in! the! 704! small!intestine,!while!the!levels!of!propionate!and!butyrate!are!higher!in!the!colon!(84)! 705! Such!a!control!has!been!described!in!Salmonella#typhimurium;!formate!acts!as!a!diffusible! 706! signal! to! induce! the! expression! of! invasion! genes! in! the! small! intestine,! the! site! that! is! 707! preferentially! colonized! by! this! enteropathogen,! while! butyrate! is! present! at! higher! 708! concentration! in! the! colon! and! repress! these! genes! (85,! 86)! It! is! tempting! to! speculate! 709! that!the!high!level!of!pyruvate!in!the!small!intestine!represses!the!expression!of!C.#difficile! 710! toxin!genes,!while!butyrate!mainly!present!in!the!colon!induces!toxin!synthesis.!Further! 711! biochemical!studies!will!be!necessary!to!characterize!the!signal?transduction!pathway!of! 712! the!CD2602?CD2601!TCS.!Thus,!the!ability!of!C.#difficile!to!monitor!the!pyruvate!level!to! 713! adapt!its!physiology,!metabolism!and!virulence!might!be!crucial!to!the!success!of!a!CDI! The! changing! epidemiology! of! Clostridium! difficile! 727! infections1274?1288.! 737! 5.! Govind' R,' Dupuy' B.' 2012.! Secretion! of! Clostridium! difficile! toxins! A! and! B! 738! requires!the!holin?like!protein!TcdE.!PLoS!Pathog!8:e1002727.! 739! 6.! Akerlund' T,' Svenungsson' B,' Lagergren' A,' Burman' LG.' 2006.! Correlation! of! 740! disease! severity! with! fecal! toxin! levels! in! patients! with! Clostridium# difficile? 741! associated!diarrhea!and!distribution!of!PCR!ribotypes!and!toxin!yields!in!vitro!of! 742! corresponding!isolates, Toxins,! 757! Butyric!Acid,!and!Other!Short?Chain!Fatty!Acids!Are!Coordinately!Expressed!and! 758! Down?Regulated! by! Cysteine! in! Clostridium# difficile.! Infection! and! Immunity! 759, 1998.
, Martin<Verstraete'I.'2011.!The!key! 765! sigma! factor! of! transition! phase,! SigH,! controls! sporulation,! metabolism! and! 766! virulence! factor! expression! in! Clostridium! difficile! Characterization! of! the! sporulation! initiation! 770! pathway! of! Clostridium# difficile# and! its! role! in! toxin! production! Integration! of! metabolism! and! 773! virulence!by!Clostridium#difficile!CodY.!J!Bacteriol!192:5350?5362.! 774! 18 775! DA,' Martin<Verstraete' I,' Dupuy' B.' 2012.! Global! transcriptional! control! by! 776! glucose! and! carbon! regulator! CcpA! in! Clostridium! difficile.! Nucleic! Acids! Res! 777! 40:10701?10718.! 778! 19!Antibiotic?induced!shifts!in!the!mouse!gut! 780! microbiome! and! metabolome! increase! susceptibility! to! Clostridium! difficile! 781! infection Martin< 783! Verstraete'I.'2010.!Global!regulation!of!gene!expression!in!response!to!cysteine! 784! availability!in!Clostridium#perfringens J,' 789! Randazzo' F,' Grandi' G.' 2002.! Gene! expression! profile! in! Neisseria# meningitidis! 790! and! Neisseria# lactamica! upon! host?cell! contact:! from! basic! research! to! vaccine! 791! development! Hatzios' SK,' Bertozzi' CR.' 2011.! The! regulation! of! sulfur! metabolism! in! 793! Mycobacterium!tuberculosis, ?973.! 803! 27.! Soutourina'O,'Dubrac'S,'Poupel'O,'Msadek'T, pp.206-219, 2001.
, 812! regulation!and!metabolic!engineering! 813! 31 Martin< 814! Verstraete'I.'2007.!Conversion!of!methionine!to!cysteine!in!Bacillus#subtilis#and! 815! its!regulation187?197.! 816! 32!The!PatB!protein!of! 817! Bacillus#subtilis!is!a!C?S?lyase! 819! Biochemical! features! and! functional! implications! of! the! RNA?based! T?box! 820! regulatory!mechanism36?61.! 821! 34.! Even'S,'Burguière'P,'Auger'S,'Soutourina'O,'Danchin'A,'Martin<Verstraete'I.' 822! 2006.! Global! control! of! cysteine! metabolism! by! CymR! in! Bacillus# subtilis.! J! 823! Bacteriol!188:2184?2197.! 824! 35.! Soutourina'O,'Poupel'O,'Coppee'JY,'Danchin'A,'Msadek'T,'Martin<Verstraete' 825! I.' 2009.! CymR,! the! master! regulator! of! cysteine! metabolism! in! Staphylococcus! 826! aureus,! controls! host! sulfur! source! utilization! and! plays! a! role! in! biofilm! 827! formation.!Mol!Microbiol!73:194?211.! 828! 36.! Fagan' RP,' Fairweather' NF.' 2011.! Clostridium! difficile! has! two! parallel! and! 829! essential!Sec!secretion!systems! 831! Evaluation! of! a! quantitative! screening! method! for! hydrogen! sulfide! production! 832! by!cheese?ripening!microorganisms:!the!first!step!towards!l?cysteine!catabolism.! 83335551?35560.! 838! 39.! Heap' JT,' Pennington' OJ,' Cartman' ST,' Carter' GP,' Minton' NP.' 2007.! The! 839! ClosTron:! A! universal! gene! knock?out! system! for! the! genus! Clostridium.! J! 840! Microbiol!Methods!70:452?464.! 841! 40.! Heap'JT,'Pennington'OJ,'Cartman'ST,'Minton'NP.'2009.!A!modular!system!for! 842! Clostridium!shuttle!plasmids!Analysis!of!relative!gene!expression!data!using! 844! real?time! quantitative! PCR! and! the, Cysteine!metabolism!and!its!regulation!in! 811! bacteria Mullany' P,' Fairweather' NF,' Minton' N,' Stabler' R,' 854! Thomson'NR,'Roberts'AP,'Cerdeno<Tarraga'AM by INNSTITUT PASTEUR-CeRIS http, 1995.
, !The!molecular!evolution!of!pyridoxal?5'?phosphate? 862! dependent!enzymes129?184.! 863! 47 Martin< 864! Verstraete' I,' Soutourina' O.' 2008.! S?box! and! T?box! riboswitches! and! antisense! 865! RNA! control! a! sulfur! metabolic! operon! of! Clostridium! acetobutylicum.! Nucleic! 866! Acids!Res!36:5955?5969.! 867! 48! A! gene! encoding! L?methionine! gamma? 869! lyase! is! present! in! Enterobacteriaceae! family! genomes:! identification! and! 870! characterization! of! Citrobacter! freundii! L?methionine! gamma?lyase.! J! Bacteriol! 871! 187:3889?3893.! 872! 49.! Dias' B,' Weimer' B.' 1998.! Purification! and! characterization! of! L?methionine! 873! gamma?lyase! from! Brevibacterium# linens! BL2! Current! therapeutics,! their! problems,! and! sulfur? 876! containing?amino?acid! metabolism! as! a! novel! target! against! infections! by! 877! "amitochondriate"!protozoan!parasites2015.!Time?resolved! 886! amino! acid! uptake! of! Clostridium! difficile! 630Deltaerm! and! concomitant! 887! fermentation!product!and!toxin!formation.!BMC!Microbiol!15:281.! 888! 54.! Hullo' MF,' Auger' S,' Dassa' E,' Danchin' A,' Martin<Verstraete' I.' 2004.! The! 889! metNPQ! operon! of! Bacillus# subtilis! encodes! an! ABC! permease! transporting! 890! methionine!sulfoxide,!D?!and!L?methionine! Comparative! 892! genomics! of! the! methionine! metabolism! in! Gram?positive! bacteria:! a! variety! of! 893! regulatory!systems, Martin< 896! Verstraete' I.' 2013.! Genome?Wide! Identification! of! Regulatory! RNAs! in! the! 897! Human!Pathogen!Clostridium!difficile.!PLoS!Genet!9:e1003493.! 898! 57.! Burguiere'P,'Auger'S,'Hullo'MF, pp.779-786, 2000.
, 2012.! Effect! of! iron! 911! limitation! and! fur! gene! inactivation! on! the! transcriptional! profile! of! the! strict! 912! anaerobe!Clostridium!acetobutylicum.!Microbiology!158:1918?1929.! 913! 63! Role! of! the! 914! Fur!regulon!in!iron!transport!in!Bacillus!subtilis! Ferric! Uptake! Regulator! Fur! Control! of! Putative! 916! Iron!Acquisition!Systems!in!Clostridium!difficile 919! Dunman' PM,' Skaar' EP.' 2010.! Staphylococcus! aureus! fur! regulates! the! 920! expression!of!virulence!factors!that!contribute!to!the!pathogenesis!of!pneumonia.! 921! Infect!Immun!78:1618?1628.! 922! 66!Analysis!of!proline!reduction!in!the! 926! nosocomial!pathogen!Clostridium!difficile! Genetics! and! regulation! of! the! major! 928! enzymes!of!alanine!synthesis!in!Escherichia!coli! Regulation! of! CodY! 932! activity! through! modulation! of! intracellular! branched?chain! amino! acid! pools.! J! 933! Bacteriol!192:6357?6368.! 934! 71.! Fonknechten'N,'Chaussonnerie'S,'Tricot'S,'Lajus'A,'Andreesen'JR,'Perchat'N,' 935! Pelletier' E,' Gouyvenoux' M,' Barbe' V,' Salanoubat' M,' Le' Paslier' D,' 936! Weissenbach' J,' Cohen' GN,' Kreimeyer' A.' 2010.! Clostridium! sticklandii,! a! 937! specialist!in!amino!acid!degradation:revisiting!its!metabolism!through!its!genome! 938! sequence, 555.! 939! 72.! Troxell' B,' Hassan' HM.' 2013.! Transcriptional! regulation! by! Ferric! Uptake! 940! Regulator!(Fur)!in!pathogenic!bacteria.!Front!Cell!Infect!Microbiol!3:59.! 941! 73.! Dalet' K, pp.368-946, 1981.
, ! Comparative! analyses! imply! that! the! enigmatic! 958! Sigma! factor! 54! is! a! central! controller! of! the! bacterial! exterior.! BMC! Genomics! 959! 12:385.! 960! 79! Identification! of! a! target! gene! and! activating! 961! stimulus! for! the! YpdA/YpdB! histidine! kinase/response! regulator! system! in! 962! Escherichia!coli! 964! Extensive! exometabolome! analysis! reveals! extended! overflow! metabolism! in! 965! various!microorganisms! 967! Genome!sequence!of!Bacillus!licheniformis!WX?02.!J!Bacteriol!194:3561?3562.! 968! 82! Global! analysis! of! gene! expression! in! an! rpoN! mutant! of! Listeria! 970! monocytogenes.!Microbiology!150:1581?1590.! 971! 83 972! VB.' 2015.! Dynamics! and! establishment! of! Clostridium! difficile! infection! in! the! 973! murine!gastrointestinal!tract!Formate!acts!as! 981! a!diffusible!signal!to!induce!Salmonella!invasion' Mullany! ' Generation! of! an! erythromycin? 983! sensitive! derivative! of! Clostridium! difficile! strain! 630! (630Deltaerm)! and! 984! demonstration!that!the!conjugative!transposon!Tn916DeltaE!enters!the!genome!of! 985! this!strain!at!multiple!sites 987! Cheung,' and' J.' I.' Rood.' 2006.! Construction! and! analysis! of! chromosomal! 988! Clostridium#difficile#mutantsReconstruction'of'sulfur'metabolism'in'C.+difficile, 122.! 966! 81.! Yangtse', pp.1335-1351, 2004.
, # metK:! SAM! 1026! synthetase! (CD0130);! mtnN:! adenosylhomocysteine! nucleosidase! (CD2611);! luxS:! S? 1027! ribosylhomocysteine!lyase!(CD3598);!mdeA:#methionine!??lyase!(CD3577),!metNIQ1:!ABC? 1028! ! 31! transport!system!methionine!(CD1489?1491);!pepT 1031! ribosyl?homocysteine.! Ext! means! " external " .! As! indicated,! an! S?box! motif! is! located! 1032! upstream! of! the! metYUmetA! and! metNIQ1# operons! and! of! the! metQ2# and! metK# genes,! 1033! suggesting! that! they! are! controlled! by! a! SAM?dependent! riboswitch! (55)! A! Tbox! is! 1034! present!upstream!of!hom?CD1580.! 1035! ! 1036! Figure'4.!Hydrogen'sulfide'production'in'strain'630?erm.! 1037! A)!Detection!of!H2S!production!using!lead?acetate!paper.!H2S!production!was!evaluated!in)! media.! The! production! of! 1039! H2S!yielded!a!black!color!due!to!the!formation!of!PbS.!B)!Detection!of!the!homocysteine!g? 1040! lyase!activity!on!a!zymogram.!Crude!extracts!of!strain!630?erm!grown!in!PY,!PYC!or!PYHC! 1041! were! loaded! on! a! native! polyacrylamide! gel! (12! %)! and! incubated! with! 10! mM! 1042! homocysteine.!Homocysteine!??lyase!was!detected!by!the!formation!of!insoluble!PbS!via! 1043! the!release!of!H2S.!Lanes!of!the!zymogram!have!been!reorganized!from!the!same!image!to! 1044! present!data!chronologically.!C)!Quantitative!detection!of!H2S!after!6!h!or!10!h!of!growth! 1045! of! strain! 630?erm! in! PY! (white! boxes)! or! PYC! (black! boxes).! H2S! production! was! 1046! measured! using! the! quantitative! methylene! blue! method,! as! described! in! the! materials! 1047! and!methods!section.!The!statistical!analysis!was!performed!by!using!Mann?Whitney!test! 1048! for! all! genes!Samples!were!charged!on!a!native! 1052! polyacrylamide! gel! (12! %)! and! incubated! with! 10! mM! cysteine.! The! cysteine! 1053! desulfhydrases!were!detected!by!the!formation!of!insoluble!PbS!formed!by!the!release!of! 1054! H2S.!The!results!presented!are!representative!of!at!least!three!independent!experiments!Analysis'of'Fur<regulated'genes'induced'in'the'presence'of'cysteine! 1059! A)!Consensus!sequence!of!the!Fur!box!motif!of!C.#difficile.!The!sequence!logo!was!created! 1060! by!the!alignment!of!putative!Fur?! The! height! of! the! nucleotides! is! 1062! proportional! to! their! frequencyB)! Effect! of! cysteine! on! the! transcript! level! of! fur# 1063! (CD1287),!feoB1#(CD1479),!cysK#(CD1594),!fhuD#(CD2878)!and!CD2992!in!630?erm!and! 1064! the! 630?erm::fur.! The! 630?erm! (white! boxes)! and! 630?erm::fur! (black! boxes)! strains! 1065! were! grown! for! 10! h! in! PY! or! PYC.! qRT?PCR! results! are! presented! as! the! ratio! of! the! 1066! amount!of!mRNA!(arbitrary!units)!of!each!gene!in!bacterial!cells!grown!in!PYC!to!that!of! 1067! each! mRNA! in! the! bacterial! cells! grown! in! PY.! Data! are! the! averages! of! at! least! three! 1068! independent!experiments!(error!bars!are!the!standard!deviations!from!the!mean!values).! 1069! C)! Aspect! of! the! bacterial! pellet! of! strain! 630?erm! grown! for! 10! h! in! PY! or! in! PYC.! The! 1070! black!precipitate!is!due!to!FeS!precipitation.!! 1071! ! 1072! Figure' 6.! Overview' of' C.+ difficile' genes' involved' in' carbon' and' amino' acid' 1073! metabolism'that'are'differentially'expressed'in'the'presence'of'cysteine, anaerobic! sulfite! 1022! reductase! (CD2231?2233);! ssuCBA1:! ABC?transport! system! sulfonates! (CD1482?1484);! 1023! ssuCBA2:! ABC?transport! system! sulfonates! (CD2989?2991);! metA:! homoserine! acetyl? 1024! transferase! (CD1826);! metY:! OAH! thiol?lyase! (CD1825);! malY:! cystathionine! ??lyase! 1025! (CD3029);! metH:! cobalamin?dependent! methionine! synthase! (CD3596)
, )! were! 1097! grown!for!10!h!in!PY!or!PYC.!TcdA!production!was!estimated!from!crude!extracts!by!dot? 1098! blot!analysis!using!an!anti?TcdA!antibody.!The!results!are!representative!of!at!least!three! 1099! independent!experiments.!C)!Effect!of!cysteine!on!tcdA,!tcdB!and!tcdR!transcript!levels!in! 1100! 630?erm::sigL!(white!boxes)!or!630?erm::sigL!complemented!with!pDIA6309?sigL!(black! 1101! boxes)!versus!the!wild?type!strain!630?erm.!All#strains!were!grown!for!10!h!in!PYC.!qRT? 1102! PCR!results!are!presented!as!the!ratio!between!the!amount!of!the!mRNA!(arbitrary!units)! 1103! of!each!gene!normalized!by!the!DNA!polIII!gene!in!both!630?erm::sigL!and!630?erm::sigL! 1104! complemented!with!pDIA6309?sigL!compared!to!the!mRNA!level!in!the!wild?type!strain! The! statistical! analysis! was! performed! by! 1107! using!a!t?test!for!all!genes!with!an!exception!for!tcdB#(Mann?Whitney!test).! 1108! ! 1109! Figure' 8.' Effect' of' sigL' inactivation' on' cysteine' degradation' and' pyruvate' 1110! production' 1111! A)! Detection! of! H2S! production! in! the! 630?erm,# 630?erm::sigL! and! 630?erm::sigL! +! 1112! pDIA6309?sigL! strains! grown! in! PYC! by! using! lead?acetate! papers.! B)! Quantitative! 1113! detection! of! pyruvate! in! the! supernatant! of! strains! 630?erm,# 630?erm::sigL! and! 1114! 630?erm::sigL! +! pDIA6309?sigL! after! 10! h! of! growth! in! PY! (white! boxes)! or! PYC! (black! 1115! boxes).!The!statistical!analysis!was!performed!by!using!Mann?Whitney!test!for!all!genes,! 1116! ns:!non!significant.! 1117! ! 1118! Figure'9.'Effect'of'pyruvate'and'Na2S'on'toxin<gene'expression' 1119! A)!Transcript!levels!of!tcdA, ccpA# (A)! and! strains! 1096! 630?erm