Ecoli Citrate Metabolism Evolution_STD

8/18/2019 Ecoli Citrate Metabolism Evolution_STD

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The Case ofCitrate Metabolism

Evolution in E. coliBacteria

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2ntroduction

These slides are provided as a teachin& resourcefor the E. coli citrate metabolism case as describedon www.evo-ed.com. fuller description of thecase can be found on the website.

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Bac6&round:

Citrate Metabolism in E.coli Bacteria


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Bacteria Basics

Bacteria are considered one of theearliest forms of life.

%ound an!where and

ever!where: animal &uts*oceanic h!drothermal vents*even radioactive waste

Estimated that there are D 10,0 bacterial cells on Earth

The human bod! harbors upwards of 1000

di;erent bacteria species


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Hiversit! in (hapes

occu% bacillu

ibripirochete


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E. coli basics:

%ull name: Escherichia coli(hape: Bacillus

(iIe: $ Jm K 0. Jm

%ound in the di&estive tractsof most warm-bloodedanimals

Lenerall! harmless e3cept

for a few strains that cancause foodborne illnessFe.&. E. coli 1N:9NG

ne of the most studied and well-characteriIed

or&anisms in e3istence.


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"h!lo&enetic Tree of )ife


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Escherichia coli is a

proteobacterium

• "roteobacteria is a ma#orph!lum within the bacterialdomain.

•ll proteobacteria are &ram-ne&ative* that is* the! have athin peptido&l!can la!erbetween the cell membraneand an outer membrane.

• E. coli is a member of theclass &ammaproteobacteria.


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9ow is E. coli transmitted amon&speciesO

Gansheroff L J , and O'Brien A D PNAS 2000;97:2959-29!

©2000 by National Academy of Sciences


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E. coli >esearch is Common

• Eas! to &row and maintain - can &rowbetween PN and QRC* optimal &rowth at,NRC

• Aer! small or&anism*but &rows to form ver!lar&e populations.

• >eproduce rapidl!*with &eneration timesas low as of $0 minutes

under optimal conditions.


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E. coli +nder the MicroscopeF(cannin& Electron Micro&raphG


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E. coli +nder the MicroscopeFTransmission Electron Micro&raphG


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The )on&-Term Evolution

E3periment on E. coli


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The )on&-Term Evolution E3periment

• Be&an in %ebruar! of 1QSS b! Hr.>ichard )ens6i usin& E. coli tostud! evolution in action.

• Aer! simple idea: &row E. coli inserial broth cultures for a lon&time and see what happens.

• Cultures have been &rowin&

nearl! ever! da! since* resultin&in over 0*000 &enerations of&rowth and countin& Feuivalentto over 1 million !ears of human

evolutionG

Hr. >ichard )ens6i*Histin&uished "rofessor of

Microbial Ecolo&!* Michi&an

(tate +niversit!


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• The broth contains a small amount of&lucose for the bacteria to use as afood source.

• nother possible food source* citrate,is also present in the broth* but thebacteria cannot &row on it under the

conditions of the e3periment.

Llucose Citrate


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9ow does evolution happen in the

e3perimentO

• Hr. )ens6i be&an twelve* initiall! identical populations*each in its own Uas6?.

• The populations are 6ept completel! isolated from oneanother* preventin& an! &ene Uow between them.


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E3perimental "rotocol


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• The populations have beentransferred almost ever! da! since*evolvin& for over 0*000 &enerations

and countin& Feuivalent to over 1.$million !ears of human evolutionG


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1 !ears into the e3periment*somethin& ver! une3pected

happenedV


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i%cu%%ion ue%tionh! is population WQ cloudier than

the othersO


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The &rowin& mediumV

• >emember* the bacteria &row in amedium that contain% both &lucose andcitrate molecules.

• 'ormall!* E. coli cannot use citrate in thee3perimental environment.

• Could the bacteriain Uas6 WQ haveevolved the abilit!to &row on citrateO


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Citrate vs Llucose


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9ow does evolution happen in the

e3perimentO

• Evolution occurs b! mutation and natural %election.

• +utation is when a chan&e in the H' seuence occurs in

an individual. This chan&e ma! or ma! not a;ect a trait* and

ma! have a neutral* benecial* or detrimental e;ect.• Natural %election is a process in which or&anisms with

favorable traits are better able to survive and reproduce* and

are therefore more li6el! to pass on their traits to the ne3t

&eneration. (imilarl!* or&anisms with detrimental traits are

less able to survive and reproduce* and are therefore less

li6el! to pass on their traits to the ne3t &eneration.

Conseuentl!* over time the population becomes better able

to survive and reproduce in the environment in which it lives


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Cell Biolo&! of CitrateMetabolism in E. coli

Bacteria


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erobic Citrate Metabolism Evolves

• fter P,,*000 &enerations F1 !earsinto the e3perimentG* population WQwas observed to be cloudier than an!

other population. – This means there was a si&nicant

increase in bacterial &rowth in

population WQ.


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erobic Citrate Metabolism Evolves

• 2nvesti&ations indicated that the cells inpopulation WQ were able to import citratefrom the medium.

• This is unusual &iven that E. coli &enerall!cannot import citrate in the o3ic FaerobicGconditions present in the e3periment.

• nce citrate enters the E. coli cell* it canbe metaboliIed in Citric cid c!clereactions.


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2n the broth* the ener&!molecule used b! thebacteria is $luco%e.

second ener&!molecule called citrate

is also present in the brothbut it can onl! bemetaboliIed in the absenceof o3!&en.


Llucose

Citrate


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9ow is Citrate used forener&!O

• Citrate is important in biolo&!* as it isan intermediate in the citric acidc!cle

– Citric cid c!cle &enerates cellularener&! in all aerobic or&anisms F!es*even humansG

– hen imported* citrate is incorporatedinto the Citric cid c!cle


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Citrate vs Llucose

The nutrient broth contains morecitrate F1N00 JMG than &lucose F1,Q

JMG.


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Citrate vs Llucose

The transport of citrate into the E coli cell isnot possible when o3!&en is present in the

environment.


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Citrate vs Llucose

2f citrate could &et into the cell* the bacteriumcould metaboliIe it in Citric cid c!cle reactions*resultin& in a si&nicant increase in the ener&!

available.


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The CitT Transport "rotein

• The CitT protein Fin&reenG is an antiporter *meanin& that it can

transport citratemolecules into thecell in e3chan&e forsuccinate molecules.

• 2t operates viapassive transport.


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The CitT Transport "rotein

• 2n )ens6iXs E. colievolutione3periments* the

cells in populationWQ evolved a wa! toproduce the CitTtransport protein in

o3ic FaerobicGconditions.


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2n )ens6iXs E. coli evolutione3periments* the cells inpopulation WQ evolved a wa!to produce the CitT transportprotein in o3ic conditions.

CitT Transport "rotein Evolution


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This allows them to importcitrate into the citric acidc!cle* &ain 1T" and $'H9from it* and e3port it as

succinate in return for morecitrate.

CitT Transport "rotein Evolution


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dvanta&e of E3chan&in& (uccinate

for CitrateO

• Citrate has more potential ener&!than succinate:

– Citrate !ields 'H9* 'H9* T"

– (uccinate !ields %H9$* 'H9

• B! e3portin& succinate to importmore citrate* the second half of the

Citric cid c!cle is b!passed and therst half can be repeated.


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More available ener&! results inmore &rowth* and ultimatel! a

denser culture with a hi&herpopulation of E. coli.


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• The abundance of citrate was a lar&epotential food source waitin& to bee3ploited until &enerations when the

abilit! to transport citrate into thecell evolved.

Conseuences of CitrateMetabolism


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>eview

1. 9ow is citrate used as a source ofener&!O

$. 9ow is citrate imported into the cellO

9ow is succinate involvedO

,. hat is uniue about the cells fromWQO

. here does the citrate come fromOhere does the succinate &oO


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ptional Cellular >espiration

Calculations:

1. 9ow man! moles of T" can be made permole of &lucoseO

$. 9ow man! moles of T" can be made permole of citrateO

,. 9ow man! moles of T" can be made permole of succinateO

. Calculate the percent increase in ener&!

stores if E coli trades out succinate for citrate.ssume that the above reactants are full! o3idiIed too3aloacetate and that 'H9/%H9$ molecules are used to

build up a proton &radient for o3idative phosphor!lation.


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The Molecular Lenetics of CitrateMetabolism in E. coli Bacteria


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Bacterial Lenetics Terms and

Henitions

• /peron: Cluster of &enes under the re&ulator!control of a promoter.

• romoter%: H' seuences that bind >'pol!merase and transcription factors."romoters initiate transcription Fturn on &enesGfor production of m>'< usuall! locatedupstream of the &ene it controls.

• /perator%: re&ions of H' associated withpromoters that bind re&ulator! proteins toeither promote or hinder >' pol!merasebindin& to promoter.


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The cit operon

• The citrate-succinate transporter&ene* citT is a &ene within the cit operon.


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The cit operon

• The &enes of the cit operon aretranscribed from a sin&le promoterlocated at the be&innin& of the operon.

• The transcribed m>' FbottomG is thentranslated b! a ribosome into proteins.


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The cit operon

• %or simplicit!Xs sa6e* we willrepresent the cit operon as picturedbelow.


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• 2f citT is not transcribed* the CitTtransport protein cannot be made* andE. coli cannot transport environmentalcitrate into the cell.

Central Ho&ma of protein s!nthesis

Transcription is stopped b! ne&ativecontrol

'e&ative control of the cit operon

1'N' Dtranscription m!NA Dtranslation !/2'N

1'N' Dtranscription m!NA Dtranslation !/2'N


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>epressor protein

isactivate

d b!

• n the pre%ence of o",$en arepressor protein binds to the citpromoter and bloc6s transcription.

• Therefore* when E. coli is in an aerobic

environment* the &enes in the cit operon* includin& citT, are nottranscribed.


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Lene nottranscribedtransport protein

>epress

orprotein

• n the pre%ence of o",$en arepressor protein binds to the citpromoter and bloc6s transcription.

• Therefore* when E. coli is in an aerobic

environment* the &enes in the cit operon* includin& citT, are nottranscribed.


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• stretch of H' in the re&ion of thecitT &ene was duplicated.

• This mutation occurred randoml! withinE. coli population WQ. 2t has not* to our

6nowled&e* occurred in an! of the other11 E. coli populations.

Lenetic mutation


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• The duplication chan&ed how the &enesand promoters in this re&ion of H'were arran&ed.

Lenetic mutation


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• s a result of the duplication event* the&enes and promoters in this re&ion ofH' were rearran&ed.

Lenes and promoters rearran&ed

newarran&ement


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• 2n o3ic conditions* the cit operonpromoter is still inhibited b! a repressorprotein.

'ew Behavior in 3ic Conditions

repressor

'ew Behavior in 3ic


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• 2n o3ic conditions* the cit operonpromoter is still inhibited b! a repressorprotein.

• The &enes in the cit operon* includin&

the ori&inal cop! of citT are nottranscribed.

'ew Behavior in 3icConditions

repressor


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• 2n o3ic conditions* the cit operon promoteris still inhibited b! a repressor protein.

• The &enes in the cit operon* includin& theori&inal cop! of citT are not transcribed.

• 9owever* the promoters downstream ofthe cit operon are not Fand never wereGrepressed b! o3!&en.


repressor


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repressor

• 2n o3ic conditions* the cit operon promoteris still inhibited b! a repressor protein.

• The &enes in the cit operon* includin& theori&inal cop! of citT are not transcribed.

• 9owever* the promoters downstream ofthe cit operon are not Fand never wereGa;ected b! o3!&en.


• The repositioned rnk promoterfacilitates the transcription of the

downstream H'* includin& the


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• cit T is now transcribed and translatedinto the citrate-succinate transporter.

• Bacteria that can ma6e this proteinhave an advanta&e over those that

cannot because the! can transportener&! rich molecules into the cell.

Citrate-(uccinate Transporter

Lene is transcribed and them>' is translated<

transmembrane protein is

repressor


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The Ecolo&! and "h!lo&enetics of

Citrate Metabolism in E. coli Bacteria


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Ecolo&! of %las6 WQ

The bacterial population in %las6 WQ thatevolved the abilit! to e3press the citratetransporter in aerobic conditions is called CitY

CitY was not the onl! population in %las6 WQ:the predecessor population* called Cit-* was

still there.

These two populations* in the environment of

the Uas6* created a little ecos!stem that can bestudied.


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Ecolo&! of %las6 WQ

ne mi&ht e3pect CitY to ta6e over uic6l!due to the abundance of citrate available*but the two strains developed two di;erent

niche%* so the! were able to coe3ist.

Ba%ic ecolo$ical principle: for twoor&anisms to coe3ist in the sameenvironment* the! need to e3ploit twodi;erent niches.


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hich 'iches HevelopedO

>ecall that the CitT protein exchanges citrate for succinate Fcitrate comes inand succinate &oes outG

fter CitY evolved* a pool of %uccinateaccumulated in the environment assuccinate moved out of the CitY cellsin e3chan&e for citrate.

ithin the e3perimental populations* E. coli cellsFboth Cit- and CitYG have the abilit! to &row onsuccinate* so there were now three di;erent carboncompounds available in the environment: $luco%e4

citrate4 and %uccinate

The %las6 WQ ecos!stem


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The %las6 WQ ecos!stem*

before CitY evolves

1luco%e Citrate

Cit-



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The %las6 WQ ecos!stem*

#ust after CitY evolves

"i#$

1luco%e Citrate

Cit-



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The %las6 WQ ecos!stem*some time after CitY evolves

"i#$

Succinat

e

1luco%e Citrate

Cit-


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"h!lo&enetics Terms and Henitions

• h,lo$enetic%: the stud! of the evolutionar!relationships between &roups of or&anisms

• These relationships are determined b!comparin& H' seuence data for theor&anisms under stud!

• h,lo$enetic tree% are used to show theserelationships in a visual wa!

• Clade% are &roups of closel!-relatedor&anisms that share a common ancestor*which is represented as a node on the tree


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"opulation WQ "h!lo&enetics

• Entire population was hetero&eneous formuch of its histor! – "ossibl! indicative of more comple3 ecolo&ical

interaction within the population thanpreviousl! thou&ht

• , di;erent clades coe3isted with oneanother for at least 10*000 &enerations

prior to the evolution of CitY• CitY forms a fourth clade around ,,*$$$

&enerations

• This &ure is a


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ph!lo&en! of %las6WQ up to 0*000&enerations of

evolution. 2t was&enerated b! usin&whole-&enome H'seuencin& andcomparin& seuenceof individuals withinthe population.Clades 1-, are Cit-*with Clade bein&the CitY linea&e.•Branches that have

stopped areindicative of thatlinea&e becomin&rare or e3tinct.•'ote that this &ure

onl! shows data up to


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dvanced (tud!

>ead the followin& article and ma6e alist of uestions to brin& to class:

Blount* Z. H.* 8. E. Barric6* C. 8. Havidson*and >. E. )ens6i. $01$. Lenomic anal!sis ofa 6e! innovation in an e3perimental

Escherichia coli population. Nature 567:1,-1S. FbstractG

http://www.nature.com/nature/journal/v489/n7417/full/nature11514.htmlhttp://www.nature.com/nature/journal/v489/n7417/full/nature11514.htmlhttp://www.nature.com/nature/journal/v489/n7417/full/nature11514.html


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Clic6er [uestion 1

• 'ormall!* can E. coli metaboliIecitrateO

. es* in conditions with o3!&en

B. es* in conditions without o3!&enC. es* in conditions with or without

o3!&en

H. 'o* not in an! conditions


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Clic6er [uestion $

• 2n conditions without o3!&en* howdoes E. coli brin& citrate into the cellO

. Citrate enters the cell via active

transportB. Citrate di;uses freel! across the cell

membrane

C. Citrate is brou&ht in throu&h anantiporter protein in e3chan&e forsuccinate

H. Tric6 uestion* E. coli canXt utiliIe

citrate without o3!&en


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Clic6er [uestion ,

• 2n the )on&-Term EvolutionE3periment* prior to the evolution ofCitY* what are the available carbon

sources in the nutrient brothO. Llucose

B. Lalactose

C. CitrateH. mmonium

E. Both and C

li 6 i


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Clic6er [uestion

• Hurin& the )on&-Term EvolutionE3periment* what interestin&phenot!pe evolved after P,,*000

&enerationsO. The E. coli evolved virulence and are

now able to infect people

B. The E. coli evolved the abilit! to utiliIecitrate in the presence of o3!&en

C. The E. coli evolved multicellularit!

H. 'one of the above

Cli 6 [ i


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Clic6er [uestion

• To &et citrate into the cell* the E. coli in the )TEE e3perienced what t!pe ofmutation that allowed them to

e3press CitT in the presence ofo3!&enO

. "oint mutation

B. Chromosomal 2nversionC. Heletion

H. Lene Huplication

Cli 6 [ i


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Clic6er [uestion

• fter the &ene duplication event* what was the&enetic basis for citrate &ettin& into the cell in thepresence of o3!&enO

A. citT was inserted into the E. coli &enome allowin& CitT

to be translated* allowin& citrate into the cellB. h!brid &ene was &enerated* which brin&s citrate

into the cell

C. plasmid was ta6en up from the environment withthe &enes reuired for citrate metabolism

D. citT was placed under the control of another promoterwhich is active in the presence of o3!&en* allowin&CitT to be produced when normall! it would not be

Cli 6 [ ti N


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Clic6er [uestion N

• h! is it practical for the CitY cells to e3portsuccinate in e3chan&e for importin& citrateO

. (uccinate cannot be metaboliIed further b! E.coli* so the cells &et rid of it

B. More ener&! can be acuired from citrate thansuccinate* so metaboliIin& citrate over succinateis more ener&eticall!-favorable for the cell

C. Citrate can be fermented b! the cells for ener&!

while succinate cannotH. 2mportin& citrate allows the cells to attain more

&lucose for ener&!

Ecoli Citrate Metabolism Evolution_STD

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