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  0           INTERACADEMIES SYMPOSIUM Académie des sciences, German National Academy of Sciences Leopoldina and The Royal Society on “The New Microbiology” May 14, 15 and 16, 2012 Institut de France 23 quai de Conti Paris 6e POSTER BOOK Organizing Committee Pascale Cossart and Philippe Sansonetti, Académie des sciences, Joerg Hacker and Juergen Heesemann, German National Academy of Sciences Leopoldina David Holden and Richard Moxon,

  •  and  norophthalmate

  •  bacterial  l-??forms

  • vacv   containing

  • ??l-?? glutamyl-??l-??cysteinyl-??glycine

  • n1  

  •  rods  to

  •  of  the


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INTERACADEMIES SYMPOSIUM
Académie des sciences, German National Academy of Sciences
Leopoldina and The Royal Society

on
“The New Microbiology”

May 14, 15 and 16, 2012

Institut de France
23 quai de Conti
eParis 6

POSTER
BOOK
Organizing Committee

Pascale Cossart and Philippe Sansonetti,
Académie des sciences,
Joerg Hacker and Juergen Heesemann,
German National Academy of Sciences Leopoldina
David Holden and Richard Moxon,
The Royal Society 1

InhibitionofapoptosisandNF-κBactivationbyvacciniavirusproteinN1occurvia
distinctbindingsurfacesandmakedifferentcontributionstovirulence

1 1 1CarlosMaluquerdeMotes ,SamanthaCooray ,HongweiRen ,GabrielMF.
1 1 2 2,3Almeida ,KeiranMcGourty ,MohammadWBahar ,DavidIStuart ,JonathanM
2 2 1Grimes ,StephenC.Graham ,GeoffreyL.Smith

1DepartmentofVirology,FacultyofMedicine,ImperialCollegeLondon,St.Mary’s
Campus,London,W21PG,UK
2TheDivisionofStructuralBiology,WellcomeTrustCentreforHumanGenetics,
UniversityofOxford,RooseveltDrive,Oxford,OX37BN,UK
3ScienceDivision,DiamondLightSourceLtd.,DiamondHouse,HarwellScienceand
InnovationCampus,DidcotOX110DE,UK

Vacciniavirus(VACV)proteinN1isanintracellularvirulencefactorthatbelongstoa
familyofVACVB-celllymphoma(Bcl)-2-likeproteins.Membersofthisfamilyinhibit
apoptosisoractivationofpro-inflammatorytranscriptionfactors,suchasinterferon
(IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Remarkably, N1
inhibitsbothapoptosisandNF-κBactivation.

TounderstandhowN1exertsthesedifferentfunctions,wehavemutatedresiduesin
theBcl-2-likesurfacegrooveandattheinterfaceusedtoformN1homodimers.
IntroductionofbulkyresiduesintheBcl-2-likegrooveabolishedonlytheN1anti-
apoptoticactivity,anditsinteractionwithcellularpro-apoptoticBcl-2proteinssuch
asBadorBid.Proteincrystallographyshowedthesemutantsdifferedfromwild-type
N1onlyatthesiteofmutation.Conversely,mutagenesisofthedimerinterface
converted N1 to a monomer and affected only inhibition of NF-κB activation.
Collectively,thesedatashowthatN1inhibitspro-inflammatoryandpro-apoptotic
signallingusingindependentsurfacesoftheprotein.

Todeterminetherelativecontributionofeachactivitytovirusvirulence,mutantN1
alleleswereintroducedintoaVACVstrainlackingN1andthevirulenceofthese
viruseswasanalysedafterintradermalandintranasalinoculationinmice.Inboth
models, VACV containing a mutant N1 unable to inhibit apoptosis had similar
virulencetowild-typevirus,whereasVACVcontainingamutantN1impairedforNF-
κBinhibitioninducedanattenuatedinfectionsimilartothatoftheN1-deletedvirus.
Thisindicatesthatanti-apoptoticactivityofN1doesnotdrivevirulenceinthesein
vivomodels,andhighlightstheimportanceofpro-inflammatorysignallinginthe
immuneresponseagainstviralinfections. 2
Ergothioneinandtheglutathioneanalogsophthalmateandnorophthalmate:from
mammalstocyanobacteria

NarainsamyK.(1),FarciS.(1),BraunE.(1),JunotC.(2),Cassier-ChauvatC.(1)and
ChauvatF.(1)
CEA,iBiTec-S(1)UMR8221,LBBCand(2)SPI,LEMM,CEASaclay91191GifsurYvette

Cyanobacteria,theonlyknownprokaryotesthatperformoxygenicphotosynthesis,
areregardedasbeing(i)amongsttheoldestlifeformsonearth;(ii)theproducersof
theEarth’soxygenicatmosphere;and(iii)theprogenitoroftheplantchloroplast.
Overtime,cyanobacteriahaveevolvedasthemostdiversegroupofbacteriathat
colonizemostbiotopes(fresh,brackishandmarinewaters,andsoils).Thehardiness
ofcyanobacteriaisduetotheirefficientphotosynthesisthatusesnature'smost
abundantresources,solarenergy,water,CO2andmineralnutrients,toproducea
largepartoftheatmosphericoxygenandorganicassimilatesforthefoodchain.
Cyanobacteriafixannuallyabout25GigatonsofcarbonfromCO2intoenergydense
biomass.Forthis,cyanobacteriauseabout0.3%ofthesolarenergy,178,000TW,
reachingtheEarthsurface,avalueexceedingbymorethan25timestheenergy
demand of the human society (about 15 TW). Furthermore, the availability of
moleculartoolsforgenemanipulationmakecyanobacteriapromising“low-cost”
microbialcellfactoriesforthecarbon-neutralproductionofbiofuels,whilesaving
arablesoilsforcrops.
Inproducingtheoxygenicatmosphere,cyanobacteriawerethefirstorganismsto
faceoxidativestressandironlimitation(duetoironoxydation),whichisespecially
detrimental to the iron-requiring photosynthesis pathway. Consequently,
cyanobacteria have developed powerful strategies to cope with the oxidant
byproductsgeneratedbytheiractivephotosynthesisandrespiration.Manyofthese
strategieswereconservedbyevolutionsuchastheproductionofglutathione(γ-L-
glutamyl-L-cysteinyl-glycine),whichwasregardedmerelyasaredoxbufferuntilwe
andothergroupsshowedthatitalsooperatesintheassemblyoftheiron-sulfur
clusterofanti-oxidantglutaredoxinenzymes.
Using a systems biology approach* (functional genomics, transcriptomics,
metabolomicsetc;..)wethoroughlyanalyzedoftheroleofglutathionesynthesis
(GshAandGshBenzymes)anddegradation(Ggt)inthephysiologyandtoleranceto
metalandoxidativestressesintwomodelcyanobacteria.
Amongotherresultswereport,forthefirsttime,thatGshAandGshBalsosynthesize
thetwoGSHanalogs,ophthalmate(γ-L-glutamyl-L-α-amino-n-butyryl-glycine)and
norophthalmate(γ-L-glutamyl-L-alanyl-glycine),whichwehadbeendescribedonlyin
mammalssofar.Wewilldiscusstheroleophthalmateandnorophthalmateinstress
signalling (ie; sulfur status or starvation) and in both amino-acid and sugars
metabolisms.
Wealsoreport,forthefirsttimethatsomecyanobacteriaareabletosynthesize
anothersulfur-containingantioxidant:ergothionein(EGT),whichwasalsodescribed
onlyinmammals,whichacquireEGTsolelythroughtheirdiet.Wewilldiscussthe
reciprocalroleofGSHinthesynthesisEGTanditsinterplayinthetoleranceto
oxidativeandmetalstresses.
*http://www.researcherid.com/rid/E-7505-2010andhttp://www.researcherid.com/rid/E-7394-2010 3
MechanismsunderlyingtheswitchfromrodstoL-formsinBacillussubtilis

PatriciaDomínguez-Cuevas,RomainMercierandJeffErrington.

CentreforBacterialCellBiology,InstituteforCellandMolecularBiosciences.
NewcastleUniversity.RichardsonRoad,NE24AX.NewcastleuponTyne.UK

The cell wall is a crucial protective layer that surrounds virtually all bacteria.
However,somebacteriahavedevelopedmechanismstoswitchbetweenwalledand
wall-deficientstates.Thesecellwall-deficientbacteria,knownasL-forms,havebeen
isolatedfrommanydifferentspecies1.Theyrepresentaninterestingmodelofstudy
considering their potential involvement in antibiotic resistance and persistent
infections2.Butdespitedecadesofstudythemechanismsunderlyingcelldivisionin
theabsenceofthecellwallarepoorlyunderstood..Wehaveisolatedastrainof
BacillussubtilisthatcanquicklyandquantitativelyconvertfromthewalledtotheL-
formstate.MutationsintwodifferentgenescontributetothehighfrequencyofL-
formtransition:walR,atranscriptionalregulatorinvolvedincellwallhomeostasis;
andsepF,requiredforaccurateandefficientcelldivision.Time-lapseimagingshows
thatthemutationsactbyfacilitatingthereleaseoftheL-formfromitswalledparent
cellbutthattheyactindifferentways3.
Inapreviouswork,weshowedthatL-formdivisiondoesnotrequireFtsZorany
residualpeptidoglycansynthesis4.Infact,manyessentialproteinsforthecellwalled
form have turned out to be dispensable for L-form survival and proliferation.
Therefore,weconductedageneticscreenusingrandomtransposonmutagenesisto
identifygenesexclusivelyessentialforL-formdivision.Ourresultshaveshownthe
crucialroleofmembranelipidcompositionanditsbiophysicalpropertiesduringL-
formdivision5.
1.AllanEJ,HoischenC,GumpertJ.BacterialL-forms.AdvApplMicrobiol.2009.68:1-
39.
2. Domingue and Woody. Bacterial persistence and expression of disease. Clin
MicrobiolRev.1997Apr;10(2):320-44.

3.Domínguez-Cuevas,P.,Mercier,R.,Leaver,M.,Kawai,Y.,Errington,J.Molecular
Microbiology.2012Jan;83(1):52-66.TherodtoL-formtransitionofBacillussubtilis
islimitedbyarequirementfortheprotoplasttoescapefromthecellwallsacculus.

4.LeaverM,Domínguez-CuevasP,CoxheadJM,DanielRA,ErringtonJ.Lifewithouta
wallordivisionmachineinBacillussubtilis.Nature.2009Feb12;457(7231):849-53.
5.Mercier,R.,Domínguez-Cuevas,P.,Errington,J.Crucialroleofmembranefluidity
inproliferationofprimitivecells.CellReports.Inpress.
4
TheYvcKproteinisrequiredformorphogenesisvialocalizationofPBP1under
gluconeogenicgrowthconditionsandisregulatedbySer/Thrphosphorylationin
Bacillussubtilis.

1 1 2 1ElodieFoulquier ,FrédériquePompeo ,AlainBernadac ,CélineFreton ,Christophe
3 1 1Grangeasse ,LeonEspinosa andAnneGalinier
1 2LaboratoiredeChimieBactérienne,UMR7283and Serviced'ImagerieCellulaire,
FR3479-IMM,CNRS,Aix-MarseilleUniversité,31cheminJosephAiguier,13402
3MarseilleCedex20,France. InstitutdeBiologieetChimiedesProtéines,UMR5086,
CNRS,UniversitédeLyon,7,passageduVercors,69367Lyoncedex07,France

TheYvcKproteinwaspreviouslyshowntobedispensablewhenB.subtiliscellsare
grownonglycolyticcarbonsourcesbutessentialforgrowthandnormalshapeon
gluconeogeniccarbonsources.YvcKislocalizedasahelical-likepatterninthecell.
This localization seems independent of the actin-like protein, MreB. A YvcK
overproduction restores a normal morphology in an mreB mutant strain when
bacteria are grown on PAB medium. Reciprocally, an additional copy of mreB
restoresanormalgrowthandmorphologyinayvcKmutantstrainwhenbacteriaare
grownonagluconeogeniccarbonsourcelikegluconate.Furthermore,asalready
observedforthemreBmutant,thedeletionofthegeneencodingthepenicillin-
binding protein PBP1 restores growth and normal shape of a yvcK mutant on
gluconeogeniccarbonsources.ThePBP1isdelocalizedinanmreBmutantgrownin
theabsenceofmagnesiumandinayvcKmutantgrownongluconatemedium.
Interestingly,itsproperlocalizationcanberescuedbyYvcKoverproduction.
Recently,weobservedanewconnectionbetweencarbonmetabolismandcellshape
regulationinBacillussubtilis.TheenzymaticpropertiesYvcKareunknown,butYvcK
possessesaRossmannfold,andisanNAD(P)(H)bindingprotein.Furthermore,YvcK,
likeitsMycobacteriumtuberculosiscounterpart,isphosphorylatedonathreonine
residue. The overproduction of protein modified at the phosphorylation site
(phosphoablativeYvcK)cannotrescueanmreBmutantandPBP1isstillmislocalized.
ThismislocalizationisalsoobservedinanmreBprkCmutantthatoverproducesYvcK.
HowYvcKinfluencesthelocalizationoractivityofPBP1isunknownbut,YvcKjoins
theincreasingnumberofproteinswithanenzymaticsignaturethataffectcellular
processes.
5
AmetabolomicsapproachtostudybacterialvirulenceinCelegansrevealsdistinct
metabolicprofiles

MichaelWitting1,2,MariannaLucio1,DimitriosTziotis1,BrigitteWägele2,3,Romé
Voulhoux4,PhilippeSchmitt-Kopplin2,5,SteveGarvis4,*

1DepartmentofGenomeorientedBioinformatics,LifeandFoodScienceCenterWeihenstephan,
Technische Universität München, 85354 Freising Weihenstephan, Germany; 2 Research Unit
Analytical BioGeoChemistry, Helmholtz Zentrum München, German Research Center for
Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany; 3 Institute of
Bioinformatics and Systems Biology, Helmholtz Zentrum München, German Research Center for
EnvironmentalHealth,IngolstaedterLandstrasse1,85764Neuherberg,Germany4CentreNationalde
laRechercheScientifique,31CheminJosephAiguier,13402MarseilleCedex20,France5Department
for Chemical-Technical Analysis Research, Technische Universität München, D-85354 Freising-
Weihenstephan,Germany

Thereremainsaneedforrobustandsimpleanimalmodelsofinfectiontoallowfor
betterunderstandingofhost-microbeinteractions,bacterialpathogenesisaswellas
development of antimicrobial therapies. An attractive model is the invertebrate
nematodeCaenorhabditiselegans,nowwellrecognizedasrelevantforthestudyof
bacterialpathogenesisaswellasamodelforotherconditions.ThewormC.elegans
issusceptibletoanumberofbacterialpathogens,whichareabletokillorinducea
rangeofsymptomsofdisease.HumanpathogensalreadyknowntoaffectC.elegans
includeGram-negativebacteriasuchas,PseudomonasandSalmonellaaswellasa
numberofGram-positivesuchasEnterococcusandStaphylococcus.Many
oftheseareabletocolonizethewormintestine,andthepathogeniceffectcanbe
measuredbymarkingthedecreaseinlifespanofthenematode.Severalstudieshave
alreadybeenundertakeninwhichmutantlibrariesofabacterialpathogenhave
beentestedinthewormtoidentifycandidatevirulencegenes.C.elegansalsooffers
researchers a simpler model for analysis of the host side of pathogen-host
interactions.Geneticanalysisofthehostisrelativelystraightforward,forexample
byscreeningforwormmutantseithermoreresistantorhypersensitivetoagiven
pathogen.InthisstudywehaveusedtheC.elegansmodelcoupledwithdirect
infusion Ion cyclotron resonance Fourier transform mass spectrometry (DI-ICR-
FT/MS)toinvestigatetheuniquemetabolicphenotypesinwormsfacingspecific
stresses,likeinfectionorstarvation.Metabolomicsisananalyticaloptionforthe
globalanalysisofmetaboliteswithinabiologicalsample,providingadirectreadout
ofanorganism’smetabolicstate.Thesereadoutsarecondition-dependentandthus
changes in given metabolite concentrations mirror changes in physiological
conditions in response to stimuli. Our aim was to undertake whole worm
metabolomicsandrecovermarkersoftheinducedmetabolicchangesinCelegans
broughtonbyinteractionwithpathogens.Inthisinvestigation,weutilizethismodel
system to reveal complex metabolic phenotypes allowing clustering based upon
challenge.Weidentifygeneralendproductindicatorsofbacterialinfectionwithin
this system, as well as specific markers for each of the experimental challenge
conditions. 6

Thedefoperonoverproduced/expressedinBacillussubtilissuppressesthemta
deletion

ElsaGermain
IMMLCBCNRS31CheminJosephAiguier,13009Marseille
InBacillussubtilis,theMtaMultidrugtransporteractivatorregulatestheexpression
ofbmrandblt,whichencodetwomultidrugeffluxtransporters.Mtawasfoundhere
toplayanunexpectedroleincentralphysiologicalprocesses,sincetheabsenceof
this activator results in low levels of CpgA, a P-loop GTPase, and this effect is
suppressed by a secondary mutation, which increases the levels of CpgA and
improvesthefitnessoftheresultingcells.ThehigherlevelsofCpgAinthesecells
mostprobablyproceedsfromanincreaseoftheexpressionofthedefoperonwhich
comprisessevengenes,def,fmt,yloMandyloN,encodingapeptidedeformylase,
theN-formyltransferase,anE.coliRsmBhomologousandaproteinofunknown
functionbelongingtotheRlmNfamilyrespectively,allofwhichcertainlyorprobably
involvedinthetranslationprocess,andprpCprkCcpgA,whichencodesaSer/Thr
signaling system involved in peptidoglycan expansion or deposition. This up
expressionofthedefoperon(Pdefup)yieldshyperchainednonmotilecells.Time
lapseexperimentsshowedthatthemtamutationhasnoeffectontheON/OFF
switchgoverningcellchainingandmotilityandisreversiblelikeinwildtypecells.
However,mtaPdefupstrainsarelockedinastateinwhichthecellseparationand
motilitygenesareintheOFFstate.TheSigDtranscriptionalfactorispresentand
activeinthemtastrain,whileitsactivityisdrasticallyreducedinthemtaPdefup
mutant,althoughtheproteinlevelisnotaffectedbythePdefupmutation.Thefact
thatIPTG-inducedPspacdefinaMta+backgroundresultsinapermanentOFFstate
showsthatanhyperchainingphenotypecouldbeobtainedwithanupexpressionof
thedefoperononly. 7
GeneticcontrolofdynamicpolarityinMyxococcusxanthus

MathildeGuzzo1*,YongZhang1,2,AnneValérieLeGall1,TâmMignot1*

1LaboratoiredeChimieBactérienne,CNRSUPR9043,InstitutdeMicrobiologiedela
Méditerranée,UniversitéAix-Marseille,31CheminJosephAiguier,13009,Marseille,
France.
2StateKeyLaboratoryofMicrobialTechnology,CollegeofLifeScience,Shandong
University,250100,Jinan,China.
*Funding:ERC-startinggrant-DOMEstg261105
Myxococcusxanthusisasocialbacteriumwithacomplexdevelopmentallifestyle.
Motile Myxococcus cells cooperate by motility and their ability to change their
directionofmovementinaprocesswherethepolesexchangeroles,allowingthe
cellstoresumeintheoppositedirection,inahighlyregulatedmanner.A
smallG-proteinMglAwasdescribedtoactivatethemotilitymachineryinM.xanthus
andtochangeitspolarlocalizationtodrivecellularreversals.ThisRas-likesmallG
proteinoscillatesbetweenactive(GTP-bound)andinactive(GDP-bound)statesto
controltheswitch.TheGTPase-ActivatingProteinMglBincreasesthehydrolysisof
GTPbyMglAtoreachitsinactivestate.Poletopoleswitchingofthesetwomotility
proteins is controlled by the Frz signal transduction pathway, a bacterial
chemosensory-like system where a soluble receptor activates a histidine kinase,
whichphosphorylatesaresponseregulatortotriggerreversals.Previously,itwas
shownthattheresponseregulatorFrzZisphosphorylatedbytheFrzkinase,however
howFrzZislinkedtotheMglABsystemhasremainedmysterious.
Inthisstudy,wearetryingtoelucidatehowtheFrzcascadeisconnectedtothe
MglA and MglB switch complex. We first found that a new response regulator
proteinRomRcontrolsthepolarlocalizationofMglAinaFrz-dependentsystem.In
thisstudy,weshowongoinggenetic,cellbiologyandbiochemicalapproachesto
elucidatehowFrzZ,RomR,MglAandMglBcooperatetoprovokethepolarityswitch.
Thisway,wehopetomodelhowexternalsignalsareintegratedtocontrolmotility
andultimatelygeneratecooperativebehaviors. 8
DevelopmentofSignatureTaggedMutagenesisofLactobacilluscaseitoidentify
genesimpliedingutpersistence.

Licandro-SerautH.(1)(2),ScornecH.(2),CavinJ.F.(2),PédronT.(1),SansonettiP.
(1).

(1)UnitédePathogénieMicrobienneMoléculaire,InstitutPasteur,Paris,France.
(2)UMRAProcédésAlimentairesetMicrobiologiques,AgroSupDijon/Universitéde
Bourgogne,Dijon,France.

Lactobacilluscaseiisawell-documentedprobioticspeciesconcerningitsbenefitfor
thehost,butmechanismsinvolvedinthesymbioticinteractionswiththehostare
poorlyunderstood.Thisisnotablyduetothelackofreliablegenetictoolstoperform
reversegeneticsinlactobacilli.Theaimofthisstudywastodevelopastrategybased
onSignatureTaggedMutagenesis(STM)(1)thatallowstheidentificationofbacterial
genesrequiredforpersistenceinthegut.Twomajorpointsweredevelopedforthis
purpose:(i)asystemforefficientrandommutagenesisinL.caseiand(ii)areliable
phenotypicscreeningmethodtodeterminetheabilityofeachmutanttomaintainin
thegut.
Atwo-plasmidsystemfortransposonmutagenesis,namedP -TpaseIS system,junc 1223
wasdesigned(2).Afirstplasmid,allowingthetransientexpressionofTpaseIS ,the1223
Lactobacillus johnsonii IS1223 transposase gene, was associated with a second
suicidetransposableplasmidcarryingthetargetsequencesofTpaseIS .Seventy1223
different tags were cloned in the second, transposable plasmid generating 70
different tagged transposable plasmids. This system gave rise to efficient
transpositionoftaggedplasmidinL.casei.Ataggedtransposonmutant
librarycomposedof60poolsof70differentmutantswasdesignedsothateach
taggedmutantofthepoolcanbequantifiedbyreal-timeqPCR.
Fourpoolsof70tagged-mutantseachwerechallengedinrabbitilealloopduring16
hourstodeterminetheirabilitytomaintaininthegut.Eachpoolwasinjectedin
loopsoftwodifferentanimals.Foreachmutant,thequantitativevariationbetween
theinoculationpoolandtherecoveredpoolwasdeterminedthankstoqPCR.Two
mutantswereshowntobealteredintheirmaintenance.Thesamemutantswere
identifiedinthetworabbits,validatingthescreeningmethod.
Thisvalidatedstrategywillbecarriedouttodeciphergenesinvolvedinprobiotics
maintenanceingutandcouldalsobeusedtostudybacterialadaptationtoother
particularenvironments.

(1)Hensel,M.,J.E.Shea,C.Gleeson,M.D.Jones,E.Dalton,andD.W.Holden.1995.
Simultaneousidentificationofbacterialvirulencegenesbynegativeselection.
Science269:400-403.
(2)Licandro-SerautH.,BrinsterS.,vandeGuchteM.,ScornecH.,MaguinM.,
SansonettiP.,CavinJ.F.,SerrorP.,DevelopmentofanefficientinvivoPjunc-
TpaseIS1223-basedsystemforrandomtransposonmutagenesisofLactobacillus
casei.Underreview(AEM00531-12).


9
Populationalandindividualadaptationofbacteria

AusselL.(1),HernándezS.(2),DucretA.(1),CasadesúsJ.(2),BarrasF.(2)

(1)CNRS,LaboratoiredeChimieBactérienne,UMR7283duCNRS,Marseille,France.
(2)DepartamentodeGenética,UniversidaddeSevilla,Seville,Spain.

Salmonellatyphimuriumisanintracellularpathogenthatcansurviveandreplicate
withinhostcells.Amongthepopulationofintracellularbacteria,theirindividual
contributiontothesuccessoftheinfectionremainslargelyunknown.Thequestion
we addressed was to understand why, among a population of bacterial cells
geneticallyidentical,someofthemhavetheabilitytoproliferateinthehostand
othersdon't.Thefirstgoalofthisworkwastostudythepopulationalheterogeneity
ofbacterialcellsunderinfectionconditions.Inafirstsetofexperiences,weanalyzed
theabilityofSalmonellatogrowinLBaftervarioustimesofinfectioninthehost:
Micemacrophageswereinfected,intracellularbacteriawerecollected,transferred
underamicroscopeina37°Cchamber,andtheirindividualviabilitywasmonitored
eitherunderagarpad(abilitytoformcolonies)orunderLBflux(measureofthe
bacterialelongation).Wehadshownthatthreehourspost-infection,animportant
partofthebacterialpopulationlostitsabilitytogrow.Moreover,amongtheviable
cells,theirdoublingtimewasextremelyvariable,suggestingadifferentmetabolic
and/orphysiologicalstatefromonebacteriumtotheother.
Recently, we used dedicated fusions to characterize the environment in which
Salmonellasurvivesandreplicates(1,2).Theexpressionlevelofeachfusionreflects
theenvironmentsensedbythebacteriuminitsvacuole:reactiveoxygenspecies,pH,
bilesalts,magnesium...
Fusing the promoter of ahpC to the GFP, we measured the level of hydrogen
peroxide (H O ) as sensed by intracellular Salmonella and we compared the2 2
individualfluorescencelevelofeachbacteriumtotheirabilitytoelongate.Ourfirst
resultssuggestthattheabilityofbacterialcellstoproliferateisindependentofthe
levelofoxidativestressexperiencedinthehost.
FusingtheosmYgenetotheGFP,westudiedtheadaptationofSalmonellatobile
(2).ExposuretobilesaltsinducedtheexpressionoftheosmYfusioninaconsistent
manner. Bacterial cells grown in the absence of bile showed lower and more
homogeneousfluorescencelevels.However,asignificantdegreeofheterogeneity
wasobserved,indicatingthatsomecellsactivatethefusionintheabsenceofbile
salts.Hence,preadaptationtobilemayresultfromactivationofbileresistance
responsesinasubpopulationofbacterialcells.Thelatterphenomenonfitsincurrent
views indicating that phenotypic heterogeneity is common in clonal bacterial
populations,andthatgeneexpressionfluctuationscanhaveadaptivevalue.

References
1.AusselL.,ZhaoW.,HébrardM.,GuilhonA.A.,VialaJ.P.M.,HenriS.,ChassonL.,
GorvelJ.P.,BarrasF.,MéresseS.MolecularMicrobiology80(2011),628-640.

2.HernándezS.,CotaI.,DucretA.,AusselL.,CasadesúsJ.PLoSGenetics8(2012),
e1002459.