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The Case ofCitrate Metabolism
Evolution in E. coliBacteria
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bout this Case:
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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 $0 minutes
under optimal conditions.
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E. coli +nder the MicroscopeF(cannin& Electron Micro&raphG
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E. coli +nder the MicroscopeFTransmission Electron Micro&raphG
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The )on&-Term Evolution
E3periment on E. coli
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The )on&-Term Evolution E3periment
• Be&an in %ebruar! of 1QSS b! Hr.>ichard )ens6i usin& E. coli tostud! evolution in action.
• Aer! simple idea: &row E. coli inserial broth cultures for a lon&time and see what happens.
• Cultures have been &rowin&
nearl! ever! da! since* resultin&in over 0*000 &enerations of&rowth and countin& Feuivalentto over 1 million !ears of human
evolutionG
Hr. >ichard )ens6i*Histin&uished "rofessor of
Microbial Ecolo&!* Michi&an
(tate +niversit!
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The )on&-Term Evolution E3periment
• The broth contains a small amount of&lucose for the bacteria to use as afood source.
• nother possible food source* citrate,is also present in the broth* but thebacteria cannot &row on it under the
conditions of the e3periment.
Llucose Citrate
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9ow does evolution happen in the
e3perimentO
• Hr. )ens6i be&an twelve* initiall! identical populations*each in its own Uas6?.
• The populations are 6ept completel! isolated from oneanother* preventin& an! &ene Uow between them.
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E3perimental "rotocol
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• The populations have beentransferred almost ever! da! since*evolvin& for over 0*000 &enerations
and countin& Feuivalent to over 1.$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.
The )on&-Term Evolution E3periment
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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'e&ative control of the cit operon
>epressor protein
isactivate
d b!
• n the pre%ence of o",$en arepressor protein binds to the citpromoter and bloc6s transcription.
• Therefore* when E. coli is in an aerobic
environment* the &enes in the cit operon* includin& citT, are nottranscribed.
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'e&ative control of the cit operon
Lene nottranscribedtransport protein
>epress
orprotein
• n the pre%ence of o",$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.
'ew Behavior in 3ic Conditions
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.
'ew Behavior in 3ic Conditions
• 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: $luco%e4
citrate4 and %uccinate
The %las6 WQ ecos!stem
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The %las6 WQ ecos!stem*
before CitY evolves
1luco%e Citrate
Cit-
The %las6 WQ ecos!stem
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The %las6 WQ ecos!stem*
#ust after CitY evolves
"i#$
1luco%e Citrate
Cit-
The %las6 WQ ecos!stem
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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&!