Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation

12 Pages·2015·1.64 MB·English

Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated
Regulation
Anne-LaureVivant,a,bDominiqueGarmyn,a,bLaurentGal,b,cAlainHartmann,a,bPascalPiveteaua,b
UniversitédeBourgogne,UMR1347Agroécologie,Dijon,Francea;INRA,UMR1347Agroécologie,Dijon,Franceb;AgroSupDijon,UMR1347Agroécologie,Dijon,Francec
Inarecentpaper,wedemonstratedthatinactivationoftheAgrsystemaffectsthepatternsofsurvivalofListeriamonocytogenes
(A.-L.Vivant,D.Garmyn,L.Gal,andP.Piveteau,FrontCellInfectMicrobiol4:160,http://dx.doi.org/10.3389/fcimb.2014.00160).In
thisstudy,weinvestigatedwhethertheAgr-mediatedresponseistriggeredduringadaptationinsoil,andwecomparedsurvival
patternsinasetof10soils.ThefateoftheparentalstrainL.monocytogenesL9(arifampin-resistantmutantofL.monocytogenes
EGD-e)andthatofa(cid:2)agrAdeletionmutantwerecomparedinacollectionof10soilmicrocosms.The(cid:2)agrAmutantdisplayed
D
significantlyreducedsurvivalinthesebioticsoilmicrocosms,anddifferentialtranscriptomeanalysesshowedlargealterationsof o
thetranscriptomewhenAgrAwasnotfunctional,whilethevariationsinthetranscriptomesbetweenthewildtypeandthe w
n
(cid:2)agrAdeletionmutantweremodestunderabioticconditions.Indeed,inbioticsoilenvironments,578protein-codinggenesand lo
anextensiverepertoireofnoncodingRNAs(ncRNAs)weredifferentiallytranscribed.Thetranscriptionofgenescodingforpro- a
d
teinsinvolvedincellenvelopeandcellularprocesses,includingthephosphotransferasesystemandABCtransporters,andpro- e
d
teinsinvolvedinresistancetoantimicrobialpeptideswasaffected.Understerilizedsoilconditions,thedifferenceswerelimited
f
to86genesand29ncRNAs.TheseresultssuggestthattheresponseregulatorAgrAoftheAgrcommunicationsystemplaysim- ro
portantrolesduringthesaprophyticlifeofL.monocytogenesinsoil. m

h
t
t
p
Listeriamonocytogenesisthecausativeagentoflisteriosis,aseri- (25).Thiswascorrelatedwithalowerlevelofexpressionofin- :/
/
ousfoodborneinfectionaffectingessentiallyimmunocompro- ternalin. a
e
mised individuals, the elderly, and pregnant women (1). The Recently,inastudyfocusingononesoil(27),weshowedthat m
pathogenislargelyspreadintheenvironment.Ithasbeenisolated theAgrsystemprovidesabenefittothepopulationsofL.mono- .a
fromwatersystems(2–4),vegetation(5),soil(6–8),farms(9–12), cytogenes, but the role of the Agr communication system in the sm
foodindustries(13–15),andthefecesofanimals(16–18).Envi- adaptationofL.monocytogenespopulationsinthenaturalenvi- .
o
ronmental adaptation requires that the cell have the ability to ronment remains poorly understood. In order to gain a better r
g
integrateenvironmentalcuesinordertoadaptitsphysiologyto understandingoftheAgr-mediatedresponseduringtheadapta- /
tion of L. monocytogenes to the telluric environment, we moni- o
the surrounding conditions through the regulation of gene ex- n
tored the fate of an inoculated L. monocytogenes parental strain
pression. Genomics showed that an important part of the L. A
andanin-frame(cid:2)agrAdeletionmutantinasetof10soilmicro- p
monocytogenesgenome(7.3%)isdedicatedtoregulationandin- r
cludes209transcriptionalregulators,15histidinekinases,and16 cosms.Wefurtherinvestigatedtheconsequencesofinactivation il
ofAgrAonthetranscriptomeofL.monocytogenesduringadapta- 1
response regulators constituting two-component systems (19). 0
tiontothesoilenvironmentthroughadifferentialtranscriptome ,
Two-componentsystemsparticipateintheabilityofbacteriato 2
analysisapproach. 0
sense and respond to fluctuating environmental conditions. 1
AgrC/AgrAisatwo-componentregulatorysystemthatispartof 9
MATERIALSANDMETHODS
b
theAgrcommunicationsystem.InitiallydescribedinStaphylococ-
Bacterialstrainsandinoculumpreparation.L.monocytogenesL9,ari- y
cusaureus,thiscommunicationsystemisorganizedasafour-gene fampin-resistant (Rifr) mutant of the parental strain L. monocytogenes g
u
operon,agrBDCA.AgrBisamembrane-boundproteinthatpro- EGD-e(28),andL.monocytogenesDG125A6,arifampin-resistantmutant e
cessesthepropeptideAgrDintoamatureautoinducingpeptide ofthe(cid:2)agrAin-framedeletionmutantDG125A(24),wereused.L.mono- s
t
(AIP).DetectionofAIPbythehistidinekinaseAgrCinducestran- cytogenesDG125A6wasisolatedonpolymyxin-acriflavine-lithiumchlo-
scriptional regulation through activation of the regulator AgrA.
DetaileddataconcerningtheroleoftheAgrsysteminthephysi-
ology of S. aureus are available (20–22). So far, its role in the Received5January2015 Accepted12May2015
adaptationofL.monocytogenestoitsenvironmentisonlypartially Acceptedmanuscriptpostedonline22May2015
understood(23).ReportsshowthattheAgrcommunicationsys- CitationVivantA-L,GarmynD,GalL,HartmannA,PiveteauP.2015.Survivalof
ListeriamonocytogenesinsoilrequiresAgrA-mediatedregulation.ApplEnviron
temofL.monocytogenesisinvolvedinadhesiontoabioticsurfaces
Microbiol81:5073–5084.doi:10.1128/AEM.04134-14.
(24)intheearlystagesofbiofilmformation(24,25)andduring
Editor:C.R.Lovell
infection of the mammalian host (25, 26). Indeed, (cid:2)agrA and
AddresscorrespondencetoPascalPiveteau,[email protected].
(cid:2)agrD in-frame deletion mutants showed defects in adherence
Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
andearlybiofilmdevelopment.Thevirulenceofa(cid:2)agrAmutant /AEM.04134-14.
was attenuated in mice, but in vitro, adhesion and invasion in Copyright©2015,AmericanSocietyforMicrobiology.AllRightsReserved.
severalcelllineswerenotaltered(26),whilevirulenceinmiceand doi:10.1128/AEM.04134-14
invasionofCaco-2intestinalcellswerereducedinanagrDmutant
August2015 Volume81 Number15 AppliedandEnvironmentalMicrobiology aem.asm.org 5073
Vivantetal.
TABLE1Nomenclature,landuse,soilparameters,andbacterialabundanceforthesoilsusedinthisstudy
Organic
content
(g·kg(cid:4)1) Cultivablebacterial
community(CFU·
Soilsampleno. Landuse Texture pH C N C/Nratio CECa WHCb(%) g(cid:4)1ofsoil[107])
748 Agriculturalfield Siltloam 8.2 9.80 1.0 9.8 13.2 18.5 5.1
749 Agriculturalfield Clayloam 8.0 19.2 1.8 10.7 16.6 24.4 5.2
750 Agriculturalfield Clay 8.0 26.4 2.8 9.4 37.8 42.3 3.1
907 Prairie Loam 5.9 25.0 2.4 10.4 11.6 20.7 4.1
909 Agriculturalfield Siltyclay 8.1 27.5 2.8 9.8 29.5 34.2 10.0
911 Prairie Siltyclay 5.9 38.1 3.7 10.3 23.6 40.7 2.6
1003 Forest Siltyclay 7.5 33.4 2.3 14.5 27.3 36.1 4.4
1005 Prairie Clay 6.7 35.3 3.9 9.1 31.4 46.6 6.0
1051 Prairie Sandyloam 5.4 26.2 2.5 10.5 8.8 18.8 4.4
1224 Agriculturalfield Clay 7.8 25.6 2.5 10.2 27.4 37.6 3.1 D
o
aCEC,cation-exchangecapacity. w
bWHC,water-holdingcapacity. n
lo
a
ride-ceftazidime-esculin-mannitol (PALCAM) agar (AES Chemunex, soil.Thevolumeoftheinoculumwasadjustedinordertoreachafinalsoil d
Bruz,France)supplementedwith200(cid:3)g·ml(cid:4)1rifampin(Sigma-Aldrich, moisturecontentof60%ofthewater-holdingcapacity(WHC).Thein- e
d
SaintQuentinFallavier,France),asdescribedbyLemunieretal.(28).This oculatedsoilswerestirredwithasterilespatula,andthenthesoilmicro-
f
Rifrmutantwasselected,asitsgrowthrateduringplanktonicgrowthand cosmswereincubatedinthedarkat25°C.Themicrocosmsweresampled ro
itsabilitytogrowasabiofilmintryptonesoybroth(TSB;AESChemunex, 5times.Thesoilswerestirredwithasterilespatulapriortosamplingof m
Bruz,France)at25°Cwithoutshakingweresimilartothoserecordedwith 1g.ThedynamicsoftheL.monocytogenespopulationsinthesoilmicro- h
L. monocytogenes DG125A. A working stock that had been stored at cosmswerefollowedimmediatelyafterinoculationandperiodicallyover tt
p
(cid:4)80°Cwasusedthroughoutthestudy.Strainsweregrownstaticallyat a14-dayperiodbyserialplatingonselectivePALCAMagar(AESChemu- :
/
25°Cfor16hin5mlofTSB.Threeindependentinoculawerepreparedby nex,Bruz,France)supplementedwith100(cid:3)g·ml(cid:4)1cycloheximideand /a
inoculating10mlofTSBtoanopticaldensityat600nm(OD )of0.04 100(cid:3)g·ml(cid:4)1rifampin(Sigma-Aldrich,SaintQuentinFallavier,France). e
600 m
andincubatingstaticallyat25°CuntiltheOD reached0.4.Thecultures RNAextractionandcDNAsynthesis.Inoculawerepreparedaspre-
600 .
werethencentrifugedat8,000(cid:5)gfor5minatroomtemperature,andthe viouslydescribed,exceptthatthepelletsweresuspendedin10mlofster- a
s
pelletsweresuspendedinNaCl(0.85%). ilizedorbioticsoilextracts.Theinoculatedsoilextractswereincubated m
Soilscharacteristicsandpreparationofmicrocosms.Tensoilsina staticallyat25°Cfor30min.RNAswereimmediatelystabilizedbytreating .
o
country-widesoilsamplingnetworkintheBurgundysectionofFrance the bacterial cultures with the RNAprotect bacterial reagent (Qiagen, r
g
weresampledonthebasisofa16-by16-kmsystematicgrid.Withina20- Courtaboeuf, France) according to the manufacturer’s instructions. /

by20-mgrid,25individualcoresamplesoftopsoil(depth,0to30cm) Threeindependentreplicateswerepreparedpercondition.Bacteriawere o
n
werecollectedwithahandaugerevery5mattheedgesofthe16inside5- harvestedbycentrifugationat8,000(cid:5)gfor5minatroomtemperature.
A
by5-msquares.Ateachsamplingsite,thecoresamplesweremixedintoa Pelletsweresuspendedin700(cid:3)lofRLTbuffer(Qiagen)supplemented p
compositesample.Soilsamplesweresievedto5mmandstoredat4°C with(cid:6)-mercaptoethanol(1%)and0.2gofRNase-freeglassbeads(diam- r
beforeanalysis.Theattributesofthesoilsamples,suchasthepedology, eter,100(cid:3)m).ThecellsweremechanicallydisruptedinaFastPrepcell il 1
chemistry,andlanduse,whichwereextractedfromtheDONESOLdata- disrupter(MPBio,IllkirchGraffenstaden,France)for3cycles(4m·s(cid:4)1, 0
base(29),andthebacterialabundancearelistedinTable1.EndogenousL. 30s).Thecellswerecentrifugedat7,000(cid:5)gfor5minat4°C,andthecell , 2
monocytogeneswasnotfoundinthesesoils.Physicalandchemicalanaly- debriswasdiscarded.Potassiumacetate(3M,pH5.5)wasaddedtothe 0
1
ses were performed by the Soil Analysis Laboratory of INRA (Arras, supernatants(10%),andthemixturewasincubatedonicefor10minand 9
France;http://www.lille.inra.fr/las).Foreachsoilsample,triplicatemi- centrifugedat14,000(cid:5)gfor5minat4°C.Theaqueousphasewasthen b
crocosmswerepreparedbyadding50gofsoiltosterile180-mlcapped precipitatedwithanequalvolumeofcoldisopropanol((cid:4)20°C),washed, y

plastictubes. anddried.Thepelletsweresuspendedin400(cid:3)lofRNase-freewaterand g
u
Sterilizedandbioticsoilextractswerepreparedformicroarrayexper- purifiedfirstonpolyvinylpolypyrrolidone(PVPP;Sigma-Aldrich,Saint e
iments.SterilizedsoilextractswerepreparedasdescribedbyPiveteauetal. Quentin Fallavier, France) columns, then on Phase Lock gel (5Prime; s
t
(30).Briefly,500gofsoilwasmixedwith750mlofwaterfor30minat120 DominiqueDutscher,Brumath,France),andfinally,onaQiagenRNeasy
rpmandautoclavedfor1hat130°C.Soilsuspensionswerecentrifugedat kit(Qiagen,Courtaboeuf,France)accordingtothemanufacturers’pro-
10,000(cid:5)gfor20min,andsupernatantswerefilteredonWhatmanpaper tocols.PurifiedRNAwasconcentratedwithaQiagenRNeasyMinElute
(Sigma-Aldrich,SaintQuentinFallavier,France).Theparticle-freesoil cleanupkit(Qiagen,Courtaboeuf,France)followingthemanufacturer’s
extract obtained was used after autoclaving (20 min, 120°C). A biotic instructions.
fractionwaspreparedbyblending100gofsoilin300mlofwaterfor1.5 cDNA strands were then synthesized using a TransPlex complete
mininaWaringblender.Toestimatethebacterialabundanceofthebiotic whole-transcriptome amplification kit (WTA2; Sigma, Saint Quentin
fraction, culturable bacterial communities were enumerated by serial Fallavier, France) according to the manufacturer’s instructions. The
platingonnutrientagar(3g·liter(cid:4)1beefextract,5g·liter(cid:4)1peptone,15 cDNAconcentrationwasdeterminedspectrophotometricallybymeasur-
g·liter(cid:4)1agar)supplementedwith100(cid:3)g·ml(cid:4)1cycloheximide(Sigma- ingthe(cid:7)at260nm((cid:7) )and(cid:7) ((cid:7) (cid:8)1.0(cid:8)50(cid:3)gDNA/ml,(cid:7) /
260 280 260 260
Aldrich, Saint Quentin Fallavier, France) to suppress fungi. Biotic soil (cid:7) (cid:9)1.8).Foreachstrainandeachincubationcondition,threesetsof
280
extractswerepreparedbyadding1mlofthebioticfractionto9mlof cDNAswerepreparedfromthreesetsofRNAsextractedfromthreeinde-
sterilizedsoilextractinordertoreachafinalconcentrationof2(cid:5)107 pendentbiologicalrepetitionsandhybridizedindependently.
cultivableCFU/ml. Whole-genomemicroarraysanddataanalysis.Acustomizedwhole-
Soilmicrocosmsurvivalassays.Fiftygramsofeachofthesoilmicro- genomemicroarrayincludingthe2,857annotatedopenreadingframes
cosmswasinoculatedtoachieveafinalconcentrationof2(cid:5)106CFU/gof (ORFs) of the genome of L. monocytogenes EGD-e was designed by
5074 aem.asm.org AppliedandEnvironmentalMicrobiology August2015 Volume81 Number15
AgrAPromotesL.monocytogenesSoilAdaptation
TABLE2Targetgenesandspecificprimersdesignedforreal-timePCR and(cid:2)agrAmutantpopulationsinsoilmicrocosms.Principalcomponents
confirmationoftheresults analysiswasusedtoinvestigatetherelationshipsbetweensoilcharacter-
Primer Sequence(5=¡3=) isticsandthedynamicsoftheL.monocytogenespopulations.Differences
insoiltexturewererepresentedbyuseoftheUnitedStatesDepartmentof
lmo0914F CATTTTACTCATTTGCGGATCAGGA
Agriculture(USDA)pedologicalclassificationsystemusingthestatistical
lmo0914R CCAAGTGCGGGCCTATTAATAAGTAAT
softwareR(v2.14.1).Then,Spearman’srankcorrelationwasusedtotest
lmo0349F TGCAGGTGGCTTTTTATTCC
thedependencebetweenthefollowingvariables:thesurvivalprofilesof
lmo0349R CAACCATTTTTCCCCACATC
both the parental and mutant strains and soil attributes (texture, pH,
lmo2257F GGGGATAAGACCCTATCATGG
organicCandNcontent,C/Nratio,andcation-exchangecapacity).
lmo2257R TGCCATTCTATTGGAAGATGG
lmo0917F TGGAGTTGCTTGCTGAAATG
RESULTS
lmo0917R CCGCTTCATTTGGTTCAGTT
L. monocytogenes population dynamics in the 10 soil micro-
lmo2711F CCACTTGTCAAGGGCAGA
lmo2711R TGCTAGACGGGGAGGT cosmsareaffectedbythedeletionofagrA.ThedynamicsofL.
agrCF2 GGGGTCAATCGCAGGTTTTG monocytogenespopulationsinmicrocosmspreparedfromsoilsfor
agrCR2 CTTTAAGTTCGTTGGTTGCCGTA which land use and edaphic parameters were different (Table 1
D
agrAF2 GCAAGCAGAAGAACGATTTCCAA andFig.1)wererecordedovera14-dayperiod.Duringincubation
o
agrAR2 CGCTGTCTCAAAAAACAAGATAT inthesesoilmicrocosms,nogrowthofeitherofthetwostrainswas w
lmo0477R GGCGAATAAAGCAGTGGGTA detectedandthepopulationsoftheparentalstrainandthe(cid:2)agrA n
lmo0477F AACCACCCATTTTCCAATCTT lo
mutant declined throughout the experiment (Fig. 2). However, a
lmo0943F CGGAAGCCCATTCTCGACT survivalpatternsdifferedaccordingtothestrainunderscrutiny. d
lmo0943R GTTACATTGTCGCCTTCTTTGTCAG e
Regardless of the soil tested, the population of the mutant was d
lmo2467F CCGGCAAGCCGTGTTTAT
reproduciblylowerthanthepopulationoftheparentalstraininall f
lmo2467R AGGTCCACTTATTGGAAAACC r
o
three replicates. Repeated-measures ANOVA showed that these
m
differencesweretransientlystatisticallysignificant.Indeed,insoil
h
samples748,749,and1224,thedifferencesweresignificantduring t
t
p
NimbleGenSystems.Atotalof13,456probeswerededicatedtothetiling thefirstweekofincubation.Insoilsamples750and911,thedif- :
/
ofintergenicregionsevery14bp.Microarrayhybridization,washes,raw ferencesweresignificantafter2daysand4daysofincubation.In /a
datapreprocessing,andnormalizationwereperformedbyPartnerchip soilsamples1005and907,theresultswerestatisticallysignificant e
m
(Evry,France)accordingtoNimbleGenstandardprotocols.DNASTAR atday2.Thesignificanceatday4wasconfirmedinsoilsamples
.
ArrayStarsoftware(Madison,WI)wasusedforanalysisofthenormalized 1003and1051.Finally,inmicrocosmspreparedwithsoilsample as
results.tstatisticsandPvalues(P(cid:10)0.05)werecalculatedtodetermine 909,thedifferenceswerestatisticallysignificantatday4andday7. m
differentiallyexpressedgenes.Thefoldchangeinthelevelsofexpression .
Thedifferencesatday14werenolongerstatisticallysignificantin o
betweenthetwostrainswascalculatedbycomparingtheexpressionlevels r
allmicrocosms. g
underthesameincubationconditions.Comparisonsbetweenbioticand /
sterilizedconditionswerealsoperformedforeachstrain.Geneswithat Interestingly,foreachstrain,theabundanceattheendofthe o
experimentwasstatisticallysignificantlydifferentaccordingtothe n
leasta2-foldchangeinexpressionwereconsideredforinterpretation.In
A
ordertoidentifytherelationshipsbetweengenesandbiologicalfunctions, p
thegeneontologywassubsequentlysearched.PvaluesandZ-scoreswere r
calculatedinordertodeterminethechancethatacertainnumberofgenes il 1
wouldbeselectedforanygivengeneontologyterm.Informationregard- 0
,
ingintergenicregions(location,presenceofnoncodingRNA[ncRNA]) 2
was retrieved from the web-accessible transcriptome browser (http: 0
1
//www.weizmann.ac.il/molgen/Sorek/listeria_browser/) provided with 9
thepublicationofWurtzeletal.(31). b
qPCR.Inordertoconfirmthefoldchangesingeneexpressionob- y

servedinthemicroarrayanalyses,thetranscriptlevelsofseveralgenes g
u
with higher and lower transcript levels were examined by quantitative e
PCR(qPCR)usingtheAbsoluteqPCRSYBRgreencarboxy-X-rhodamine s
t
(ROX)mix(ThermoScientific,DominiqueDutscher,Brumath,France).
Primers specific for each selected gene were designed (Table 2). Each
reactionmixturecontained2(cid:5)AbsoluteqPCRSYBRgreenROXmix(7.5
(cid:3)l),0.6(cid:3)MeachPCRprimer,12.5ngofDNAtemplate,andsterilewater
to15(cid:3)l.TheqPCRswerecarriedoutinaStepOnePCRsystem(Applied
Biosystems) using the following program: an initial enzyme activation
period(10minat95°C),followedby40cyclesof15sat95°Cand1minat
60°C.FollowingPCR,absolutequantificationwasperformedbyastan-
dardcurvemethod.Foreachtarget,increasingconcentrationsofL.mono-
cytogenesgenomicDNArangingfrom10to80ng/(cid:3)lwereamplifiedin
ordertodrawarelationshipbetweentheconcentrationandthenumberof
thresholdcycles.Foldchangesinexpressionwerecalculatedastheratioof
FIG1Principalcomponentanalysis(PCA)ofsoils.Thelengthanddirection
the absolute quantity of the target gene determined in the two strains
ofthelinesrepresentthecontributionofeachparameter.Thenumbersindi-
underthesameincubationconditions. catesoilsamplenumbers,andthelettersindicatewhethersoilswerefroman
Statisticalanalyses.Repeated-measuresanalysisofvariance(ANOVA) agriculturalfield(AF),aprairie(P),oraforest(F).CEC,cation-exchange
wasusedtocomparethesurvivalpatternsoftheL.monocytogenesparental capacity.
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FIG2L.monocytogenesparentalstrain(bluelines)and(cid:2)agrAmutant(redlines)dynamicsinsoilmicrocosms.Errorbarsrepresentstandarddeviationsfrom
threeindependentreplicates.*,significantdifferences(P(cid:10)0.05)wereobservedafterANOVA.
soil(Fig.3).Indeed,at14daysafterincubation,itwassignificantly 1224,wherelessthan3logunitsweredetected14daysafterinoc-
higher in soil sample 1003 (repeated-measures ANOVA, P (cid:10) ulation. The Spearman rank correlation test was used to check
0.05),whereover4logunitsofthepopulationremaineddetect- correlationsbetweenthesurvivalrateofeachstrainandthesoils’
able,thaninsoilsamples1005and1051andintherestofthesoils attributes(texture,pH,organicCandNcontents,C/Nratio,and
(soilsamples748,749,750,907,909,911,and1224).Moderate cation-exchange capacity). From these six metrics, a significant
survivaloccurredinsoilsamples1005and1051.Theweakestsur- negative correlation between the survival rate and pH was de-
vivalwasobservedinsoilsamples748,749,750,907,909,911,and tected(Spearman’s(cid:11)(cid:8)(cid:4)0.736,P(cid:10)0.05).Duringthefirstdaysof
5076 aem.asm.org AppliedandEnvironmentalMicrobiology August2015 Volume81 Number15
AgrAPromotesL.monocytogenesSoilAdaptation
FIG3L.monocytogenesparentalstrainand(cid:2)agrAmutantabundanceafter14daysofincubationinsoilmicrocosms.Errorbarsrepresentstandarddeviations
fromthreeindependentreplicates.LettersindicatethatsignificantdifferenceswereobservedafterANOVA.
D
o
w
n
theexperiment,therateofdeclinewassteeperinthesoilswithpHs acid,anddeletionofthedltoperonaffectsadhesiontocelllines lo
a
lowerthan6(soilsamples907,911,and1051).InsoilswithpHs and antimicrobial peptide resistance (32). Several are also in- d
higherthan6,thedeclineofthepopulationeitherwaslow(soil volved in the PTS system and ABC transporters. For example, e
d
samples 1003 and 1005) or became steeper only after 4 days of lmo2115 (anrB), which codes for a permease component of an
f
r
incubation(soilsamples748,749,750,909,and1224). ABCtransporter,playsaroleinnisinresistanceandalsoinbaci- o
m
Numeroustranscriptomedifferencesbetweentheparental tracin, gallidermin, and beta-lactam antibiotic resistance, and

strainandthe(cid:2)agrAmutantbackgroundareobservedinsoil lmo0153,whichcodesforaprobableABCtransporter,isaZurR- h
t
t
environments.InordertobetterunderstandtheAgrA-mediated regulatedgenewhichisinvolvedinvirulenceinthemurinemodel p
:
responseintheadaptationofL.monocytogenestothetelluricen- (33).Fromfunctionalcategory3.7(proteinsynthesis),17genes //
a
vironment,wecomparedthetranscriptomesoftheparentaland wereidentified(3.7.1,ribosomalproteinssynthesis;3.7.4,elonga- e
(cid:2)agrAstrainsduringadaptationtothesoilenvironment.Wede- tion). Finally, an important part of the differentially expressed m
.
cidedtofocusonsoilsample748,forwhichhighdifferencesin genes (87 genes) was associated with other functions, such as a
s
survival between the parental strain and the mutant were re- transposons and insertion sequences (functional category 4.4), m
corded.DeletionofagrAresultedinlargevariationsinthetran- consistedofunknownproteins(functionalcategory5.1),ordid .
o
scriptome(Fig.4).Atotalof578genesweredifferentiallytran- nothaveanysimilaritytoknowngenes. r
g
scribed.Twohundredfifty-twogeneswererecordedinthesetof In silico analysis of intergenic regions with significant differ- /
o
genes with higher transcript levels in the (cid:2)agrA mutant back- encesintranscriptlevelsbetweenthe(cid:2)agrAmutantandthepa- n

ground(seeTableS1inthesupplementalmaterial).Geneontol- rentalstrainhighlightedasetof41ncRNAs.Theirlocations,the A
ogy did not identify any functional category to be a significant flankinggenes,andthemeasuredfoldchangesinexpressionare p
r
term.Afocusonthegeneshighlyexpressedidentifiedagroupof listedinTable3.Thelevelsofthetranscriptsof10genes(therliG, il
1
122geneswithover3-foldhighertranscriptlevelsinthe(cid:2)agrA Rli47, Rli80, Rli88, Rli99, Rli106, Rli120, Rli126, Rli127, and 0
mutantbackground(seeTableS2inthesupplementalmaterial). Rli140genes)werehigherinthe(cid:2)agrAbackgroundthaninthe ,
2
Mostofthesegenescodeforfunctionsrelatedtothemetabolism parentalstrain,and31geneswereidentifiedtohavelowertran- 0
ofcarbohydrates,suchaslmo2110(mannose-6phosphateisomer- scriptlevelsinthe(cid:2)agrAbackgroundthanintheparentalstrain. 19
ase), lmo0347 (dihydroxyacetone kinase), and lmo2824 (D-3- Todate,271ncRNAshavebeenidentifiedandannotatedinthe b
y
phosphoglycerate dehydrogenase), or functions related to the genomeofL.monocytogenes(31,34–37).AmongthesencRNAs,
g
transport of proteins, such as the ABC transporter (lmo2123 to severalhavebeenlinkedtobiologicalfunctions.Theseincludethe u
e
lmo2125, lmo1739) and the phosphotransferase (PTS) system Rli28,Rli32,Rli38,andRli50genes,whichpairtomRNAswhose
s
(lmo2782,lmo2665,lmo2666,lmo2780).Fourteentranscriptional productsarepotentiallyinvolvedinbacterialadaptationandalso t
regulatorswerealsointhissetofgenes. inrodentinfection(38);theRli31andRli50genesarerequired
Moreover,328ORFswereidentifiedinthesetofgeneswith for full virulence in murine, larva, and macrophage infection
lowertranscriptlevelsinthe(cid:2)agrAmutantbackground(seeTable models (37). Moreover, the S-adenosylmethionine riboswitch
S1 in the supplemental material). As expected, lmo0048 (agrB), sreB(Rli47),whichcanbeproducedasashorttranscriptandactas
lmo0049(agrD),lmo0050(agrC),andlmo0051(agrA)wereinthis atrans-regulator,negativelycontrolsPrfAexpressionbypairing
setofgenesandshowed,respectively,5,4,4,and9timeslower tothe5=untranslatedregionofthemRNA(39).Finally,Mandin
transcriptlevelsinthe(cid:2)agrAmutantbackgroundthaninthepa- etal.predictedthatthencRNArliItargetsmRNAwithbiological
rentalstrainbackground.Geneontologyshowedthatfunctional functionsrelatedtosugarmetabolismandtransport(36).
categories 1.1, 1.2, 1.8, 1.10, 3.7.1, 3.7.4, 4.4, 5.1, and 6.0 were Transcriptome variations between the parental strain and
significantterms(Fig.5).Eightygeneswereincludedincategory the(cid:2)agrAmutantarelimitedinsterilizedsoilenvironments.In
1,cellenvelopeandcellularprocess(1.1,cellwall;1.2,transport/ sterilizedsoilextracts,deletionofagrAresultedinalimitedvari-
bindingproteinsandlipoproteins;1.8,cellsurfaceproteins;and ationofthetranscriptome(Fig.6).Indeed,86genesand29inter-
1.10, transformation/competence). Among these genes, the dlt- genic regions showed significant differences in transcript levels
ABCD operon is involved in D-alanine esterification of teichoic betweenthe(cid:2)agrAmutantandtheparentalstrain.Seventy-two
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FIG4Heatmapofthesetofgeneswithsignificantdifferencesinlevelsofexpressionbetweenthe(cid:2)agrAmutantandtheparentalstrainunderbioticsoil il
1
conditions.Expressionlevelswerestandardizedsothatthoseoftheparentalstrainundersterilizedconditionsweresetasthebaseline.Genesarerepresentedin
0
rows.Theexpressionlevelsofthesegenesundersterilizedconditionsarealsopresented. ,

2
0
1
9
genes had higher transcript levels in the mutant, and the fold ofexpressionvariedby16-,6-,and5-fold,respectively.Interest- b
y
changeinexpressionofmostofthemwaslessthan3(seeTable ingly,allentriesinthissetofgenesexceptlmo1798,lmo2286,and
g
S3 in the supplemental material). Gene ontology identified lysAwererecordedinthesetofgeneswithlowertranscriptlevels u
functional categories of intermediary metabolism 2.1.1 (spe- duringgrowthofa(cid:2)agrAmutantofL.monocytogenesEGD-eat e
s
cific pathways, 18 genes) and 2.5 (metabolism of coenzymes 25°Cinrichmedium(40). t
andprostheticgroups,12genes)tobesignificanttermsforthis Thepresenceofthebioticfractionresultsinhigherlevelsof
setofgenes.Forexample,genesinvolvedinglycolyticandspe- transcriptionofmobilityandribosomalproteingenes.Inorder
cificpathways(lmo0342tolmo0351)(31)wererepresented,as toextractinformationregardingtheresponseofL.monocytogenes
weresomegenescodingforenzymesofthecobalaminsynthe- tothesoilbioticfraction,wefocusedonthegeneswithhighex-
sispathway(lmo1190,lmo1192,lmo1193,andlmo1196). pressionlevels((cid:9)9logunits)andforwhich(cid:9)3-foldchangesin
In the set of genes with lower transcript levels in the (cid:2)agrA expressionunderbioticconditionsincomparisontothelevelsof
mutant(seeTableS3inthesupplementalmaterial),mostgenes expressioninasterilizedsoilenvironmentwererecorded.Consid-
werealsodetectedinthecorrespondingsetofgenesidentifiedto eringL.monocytogenesL9,atotalof369genesmetthesecriteria.
havelowertranscriptlevelsunderbioticconditions.Asexpected, One hundred seventeen genes had higher transcript levels (see
thefourgenesoftheagroperon(agrBDCA)weredetected,andthe TableS4inthesupplementalmaterial).Ofthissetofgenes,func-
greatestfoldchangewasdetectedwithagrD(whichhada17times tionalcategories3.7.1(ribosomalproteins,16/56genes),1.5(mo-
lower level of expression). Three genes coding for putative se- bility and chemotaxis, 7/30 genes), and, to a lesser extent, 3.7.4
creted proteins (lmo0477, lmo0478, and lmo0479) were also in- (elongation,3/5genes),4.3(phage-relatedfunctions,6/48genes),
cludedinthetop10geneswithlowertranscriptlevels.Theirlevels 1.2(transport/bindingproteinsandlipoproteins,19/331genes),
5078 aem.asm.org AppliedandEnvironmentalMicrobiology August2015 Volume81 Number15
AgrAPromotesL.monocytogenesSoilAdaptation
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FIG5Functionalcategoriesidentifiedtobesignificantbygeneontologyandthepercentageofgenesfromthesetofgeneswithlowertranscriptlevelsinthe d

(cid:2)agrAmutantthaninparentalstrainL.monocytogenesL9underbioticconditions.Thevaluesinparenthesesrepresentthenumberofgeneswithsignificant fr
o
variationsintranscriptlevels/thetotalnumberofgenesinthefunctionalcategory.IS,insertionsequence. m

h
t
t
and2.3(metabolismofnucleotidesandnucleicacids,7/61genes) units.Thismeansthateachsoilsample,characterizedbyacom- p
:
were identified by ontology analysis. Interestingly, 7 regulators bination of abiotic and biotic factors, has to be considered a //
a
were in this set of genes, including agrA (3.6-fold increased ex- unique environment. Depending on environmental factors, the e
m
pression).Thelevelsoftranscriptsofthegenesfortheglobalni- chanceofL.monocytogenessurvivalishigherinsomesoilsthanin
.
trogenregulator(GlnR),theferricuptakeregulator(Fur),andthe others.Theseresultsareconsistentwiththoseofapreviousstudy a
s
pyrimidineregulatoryprotein(PyrR)alsoincreasedinthebiotic which showed that the survival of L. monocytogenes in soil de- m
environment.Inthemutantbackground,only5geneswerein- pendedonabioticproperties,inparticular,thebasiccationsatu- .o
cludedinthesetofgeneswithhighertranscriptlevelsunderbiotic rationratio,pH,andclaycontent(42).Inourstudy,pHwasthe rg
conditions(seeTableS5inthesupplementalmaterial). predominantabioticfactorhighlightedbytheSpearmanrankcor- o/
Thepresenceofabioticenvironmentresultedinlowerlevelsof relation.ItisknownthatlowpHisdetrimentaltothesurvivalofL. n

transcriptionof253genesintheparentalbackground(seeTable A
monocytogenesinsoil,butaspHisastructuringparameterforsoil
p
S6inthesupplementalmaterial).Geneontologyfailedtoidentify
microbialcommunities,itisratherdifficulttodifferentiatethe r
functionalcategoriesinthissetofgenesexceptforcategory4.3, actualdirecteffectofpHandtheindirectroleofthecomposi- il 1
phage-relatedfunctions(13/48genes).Again,thesetofgeneswith 0
tion and structure of the microbial communities on the dy-
,
lowertranscriptlevelsunderbioticconditionswasmoremodest
namicsofthepopulationsofL.monocytogenes(42).Moregen- 2
inthemutantbackground(25genes;seeTableS5inthesupple- 0
erally, because the composition and structure of the microbial 1
mentalmaterial). 9
communitiesarestructuredbythecomplexcharacteristicsofthe
PCRquantificationofselectedtranscripts.Inordertocon- b
abioticenvironment(43–45),itisdifficulttoaddresstheweightof y
firm the results of the expression profiles, 10 target genes were
asingleparameterontheoverallsurvivalofL.monocytogenesin g
selected for quantification. Four of these genes (lmo0349, u
soil.Nevertheless,thereisclearevidencethatthebioticenviron- e
lmo0914, lmo0917, and lmo2257) were in the set of genes with
s
higher transcript levels in the mutant background, 4 (lmo0050, mentisakeyplayerinthecontrolofthepopulationsofL.mono- t
cytogenes,asevidencedbycomparisonsofpopulationdynamicsin
lmo0051,lmo0477,andlmo2711)hadlowertranscriptlevelsinthe
(cid:2)agrA mutant background, and 2 (lmo0943 and lmo2467) had microcosmsofsterilizedandunsterilizedsoils(9,46,47).More-
over,inastudyofexperimentalecology,erosionofthesoilmicro-
similar trends in both the mutant and wild-type strains. qPCR
bial diversity resulted in the persistence of the population of L.
analysis confirmed that all entries displayed patterns similar to
monocytogenesinsoil(48).TheoverallfateofL.monocytogenesin
thoseobservedwiththemicroarrayresults(Table4).
soildependsonacombinationofabioticcharacteristics,themi-
DISCUSSION crobial abundance, as well as the diversity and structure of the
OneofthetrademarksofbacteriaofthespeciesListeriamonocy- microbial communities. Other components of the soil biology
togenesistheirvarietyofhabitatsandtheircapacitytoswitchfrom couldalsoaffectthepopulationdynamicsofL.monocytogenes.For
asaprophyticmodeoflifetoinfectionofanimals.Soilisoneof example,protozoansand/ornematodescouldfacilitatethesur-
thesehabitatswhereListeriamonocytogenescanbefound(41).In vival of L. monocytogenes, and in vitro experiments suggest that
this study, comparison of the population dynamics in 10 soils theymayshedthepathogenand,hence,facilitatedispersion(41).
showedthatsurvivalisdependentonthetypeofsoil.Indeed,the Onecanspeculatethatinsoilsample1003,collectedinaforest,
finalabundanceoflisterialpopulationsrangedfrom1.5to4log theabundanceofprotozoanscouldhavefacilitatedthepersistence
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TABLE3Location,flankinggenes,andfoldchangeinexpressionofthencRNAsfoundtobedifferentiallytranscribedbetweenthe(cid:2)agrAmutant
andtheparentalstrainunderbioticconditions
Position Flankinggene
Foldchangein
ncRNAgroupandgene Start Stop 5= 3= expression
ncRNAswithsignificantlyhighertranscript
levelsin(cid:2)agrAmutant
Rli26 388707 388590 lmo0360 lmo0361 5.277
Rli88 1320429 1320501 lmo1292 lmo1293 2.532
Rli127 1473829 1473701 lmo1439 lmo1440 4.109
Rli47(sreB) 2226036 2226481 lmo2141 lmo2142 3.457
Rli120 226546 226640 lmo0219 lmo0220 2.851
rliG 2386992 2386715 lmo2302 lmo2304 2.676
Rli99 2395032 2395236 lmo2320 lmo2321 4.911
Rli140 2395244 2395032 lmo2320 lmo2321 4.911
Rli106 2594785 2594561 lmo2515 lmo2516 2.309 D
Rli52 552417 552313 lmo0517 lmo0518 3.351 o
w
Rli80 787038 787254 lmo0761 lmo0762 4.516
n
lo
ncRNAswithsignificantlylowertranscript a
levelsin(cid:2)agrAmutant d
e
Rli38 1152549 1152917 lmo1115 lmo1116 2.495 d
Rli55 1198108 1198561 lmo1170 pduQ 2.686 f
r
Rli56 1199848 1199937 pduQ lmo1172 2.624 o
m
Rli40 1275794 1275547 lmo1251 lmo1252 2.062

Rli59 1702543 1702361 lmo1652 lmo1653 2.464 h
t
Rli23 172171 172268 lmo0172 2.677 tp
Rli43 1861533 1861377 inlC rplS 2.190 :/
/
Rli44 2039087 2039375 lmo1964 lmo1965 2.154 a
e
Rli94 2039375 2039079 lmo1964 lmo1965 2.154 m
Rli60 2054162 2054501 lmo1982 ilvD 2.613 .
a
Rli45 2154765 2154852 lmo2074 lmo2075 2.440 s
Rli46 2155058 2154765 lmo2074 lmo2075 2.440 m
Rli61 2275362 2275297 lmo2187 lmo2188 2.616 .o
Rli48 2361405 2361274 lmo2271 lmo2272 2.571 rg
Rli98 2361329 2361581 lmo2271 lmo2272 2.571 /
o
Rli112 2782978 2783221 lmo2709 lmo2710 3.483 n
Rli50 2783264 2782981 lmo2709 lmo2710 3.483 A
Rlii 2842199 2841962 lmo2760 lmo2761 2.437 p
Rli25 357618 357516 lmo0330 2.731 ril
Rli27 434817 434929 lmo0411 lmo0412 2.812 1
0
Rli28 507372 507141 lmo0470 lmo0471 3.364
,

Rli78 507068 507473 lmo0470 lmo0471 3.364 2
0
Rli29 507632 507450 lmo0470 lmo0471 2.742
1
Rli52 552417 552313 lmo0517 lmo0518 2.423 9

Rli31 597806 597926 lmo0558 lmo0559 2.610 b
y
Rli32 600750 600604 lmo0560 lmo0561 2.334
g
Rli33-2 708618 708860 lmo0671 lmo0672 2.501 u
Rli35 855495 855393 lmo0828 2.642 e
s
Rli36 859521 859412 lmo0829 lmo0830 2.261 t
Rli37 907526 907832 lmo0866 lmo0867 2.395
Rli53 955824 956021 lmo0918 lmo0919 2.358
of L. monocytogenes. The abundance of fungi could also impact togenestothesoilenvironment.Itisinterestingtonoticethatthe
survival(49–51). consequencesoftheinactivationoftheAgrsystemwerelimitedin
Inastudythatfocusedononesiltyloamsoil,wepreviously soilsample1003,whichwaspermissivetoL.monocytogenes.This
showedthatimpairmentoftheresponseregulatorAgrAorinac- suggeststhattheactivityoftheAgrsystemmaybecriticalwhenL.
tivation of the production of the propeptide AgrD reduced the monocytogenes has to face unfavorable conditions and when its
fitnessofL.monocytogenesinthishabitat(27).Theresultsofthe survivaliscompromised.AgrAispartofthecomplextranscrip-
present study allow the general observation that inactivation of tionalregulatorycircuitryofL.monocytogenes,andexperimental
theAgrsystemaffectsthedynamicsofL.monocytogenesinother evidencesuggeststhattheAgrAregulonincludesgenesresponsi-
kindsofsoilwithtexturesrangingfromsandyloamtoclayand bleforthetransportandmetabolismofaminoacidsandrelated
confirmthatdeletionofagrAaffectstheadaptationofL.monocy- molecules, genes responsible for motility and chemotaxis, and
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FIG6Heatmapofthesetofgeneswithsignificantdifferencesinlevelsofexpressionbetweenthe(cid:2)agrAmutantandtheparentalstrainundersterilizedsoil rg
conditions.Expressionlevelswerestandardizedsothatthoseoftheparentalstrainundersterilizedconditionsweresetasthebaseline.Genesarerepresentedin /
o
rows.Theexpressionlevelsofthesegenesunderbioticconditionsarealsopresented. n

A
alsogenesthatcodeforregulators(40).Theobservedintercon- thatAgrAactivityparticipatesintheoveralladaptationofthecell p
nectionbetweentheAgrregulonandthe(cid:12)Bregulon(40)sustains followingtheintegrationofenvironmentalcues. ril
theideathattheimpactofAgractivityismorepronouncedunder Major variations to the transcriptome were recorded in the 1
0
biotic and abiotic stress conditions. These observations suggest parentalbackgroundunderbioticconditions.Thissuggeststhat ,

2
thesensingofthebioticenvironmenttriggersarangeofphysio- 0
logical adaptations. This adaptation could be depicted as a sce- 1
TABLE4Comparisonoffoldchangeinexpressionbetweenthe(cid:2)agrA narioinwhichthecellsurfacemustberearrangedasaprotective 9
b
mutantandparentalstrainofselectedgenesinmicroarrayandqPCR action and for the development of resistance to antimicrobial y
analyses compounds.Moreover,theupregulationofseveraltransportsys- g
u
Foldchangeinexpression tems and modification of metabolic activity are most likely, as e
s
Gene qPCR Array evidencedbytheupregulationofcarbon,nitrogen,andpyrimi- t
dinemetabolismregulators.Increasedmotilitymayalsobeastrat-
Upregulated
lmo0914 2.6 6.6 egydevelopedinresponsetothebioticenvironment.Thehigher
lmo0349 4.7 21.4 leveloftranscriptionoffurprobablyreflectstheneedtocompete
lmo2257 6.3 4.0 foravailableiron,asoverexpressionofthisregulatorwasobserved
lmo0917 7.2 3.2 underiron-limitingconditions(52).Thehigherleveloftranscrip-
tionofagrAunderbioticconditionsisanotherindicationofthe
Downregulated
involvementofAgrAintheregulationcascaderequiredtoadapt
lmo2711 (cid:4)7.2 (cid:4)5.4
lmo0050(agrC) (cid:4)8.2 (cid:4)8.7 tothebioticenvironment.Infact,theinactivationofAgrAim-
lmo0051(agrA) (cid:4)6.7 (cid:4)6.4 pededtheappropriaterearrangementofthetranscriptomeinthe
lmo0477 (cid:4)37.9 (cid:4)16.0 mutantbackground.Asaconsequence,comparisonofthetran-
scriptomesoftheparentaland(cid:2)agrAmutantstrainsshowedlarge
Unchanged variationsunderbioticconditions.Theseresultssuggestthatthe
lmo0943 1.4 extensivemodificationofgeneexpressionthatL.monocytogenes
lmo2467 1.2
undergoesduringadaptationtosoilrequiresAgrAactivity.Inthis
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Vivantetal.
study,inactivationofAgrAalteredthetranscriptionofgenesre- bioticparameters,eitherdirectlyorindirectly.Theseresultshigh-
latedtocellenvelopeandcellularprocesses.ItincludedPTSsys- lightthattheadaptationofL.monocytogenestosoilrequiresre-
tems and ABC transporters dedicated to carbohydrate uptake. shapingofgeneexpressionatthetranscriptionallevel.Finally,our
Upregulationofthesegenesintheparentalbackgroundmayillus- datasuggestthat,incomplexenvironments,theregulatorAgrA
trateametabolicadaptationrequiredtouseresourcesavailablein mayhaveacentralroleintheregulationoftranscriptionandit
thesoilenvironment,wherecompetitionforsubstratesmayoccur may participate in the modulation of the large repertoire of
(53–56). Indeed, the prevalence of nutrient acquisition mecha- ncRNAs.
nismsintheonsetofsoiladaptationhasbeenreportedpreviously
ACKNOWLEDGMENTS
(30). Similarly, modification of the bacterial cell surface is re-
quiredforenvironmentaladaptationandduringgrowthandsur- ThisworkreceivedagrantfromtheRegionalCouncilofBurgundy(ref-
vivalinsoil(30,38,57–59).InactivationofAgrAcouldhavepre- erencePARIAGRALE11).
Thefundershadnoroleinthestudydesign,datacollectionandanal-
ventedappropriateadjustmentofthecellsurfacetotheconditions
ysis,thedecisiontopublish,orpreparationofthemanuscript.
oftheenvironment.Interestingly,genesknowntocodeforpro-
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