Listeria monocytogenes Differential Transcriptome Analysis Reveals Temperature-Dependent Agr ...

13 Pages·2012·1.03 MB·English

ListeriamonocytogenesDifferential Transcriptome
Analysis Reveals Temperature-Dependent Agr
Regulation and Suggests Overlaps with Other Regulons
Dominique Garmyn1,2, Yoann Augagneur1,2, Laurent Gal3,2, Anne-Laure Vivant1,2, Pascal Piveteau1,2*
1Universite´deBourgogne,UMR1347,Dijon,France,2INRA,UMR1347,Dijon,France,3AgroSupDijon,UMR1347,Dijon,France
Abstract
Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant
regulationofgeneexpression.Amongtranscriptionalregulators,AgrAispartofanauto-inductionsystem.Temperatureis
anenvironmental cuecriticalforinvivoadaptation. InordertoinvestigatehowtemperaturemayaffectAgrA-dependent
transcription,wecomparedthetranscriptomesoftheparentalstrainL.monocytogenesEGD-eanditsDagrAmutantatthe
saprophytictemperatureof25uCandinvivotemperatureof37uC.Variationsoftranscriptomewerehigherat37uCthanat
25uC. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and
pyrimidinebiosyntheticpathwaysandphage-relatedfunctions.Deregulationsresultedinagrowthadvantageat37uC,but
affected salt tolerance. Finally, our results suggest overlaps with PrfA, sB, sH and CodY regulons. These overlaps may
suggest thatthrough AgrA, Listeria monocytogenes integrates information onitsbiotic environment.
Citation: Garmyn D, Augagneur Y, Gal L, Vivant A-L, Piveteau P (2012) Listeria monocytogenes Differential Transcriptome Analysis Reveals Temperature-
DependentAgrRegulationandSuggestsOverlapswithOtherRegulons.PLoSONE7(9):e43154.doi:10.1371/journal.pone.0043154
Editor:LaurelL.Lenz,NationalJewishHealthandUniversityofColoradoSchoolofMedicine,UnitedStatesofAmerica
ReceivedApril9,2012;AcceptedJuly17,2012;PublishedSeptember14,2012
Copyright:(cid:2)2012Garmynetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Thisworkwasfundedbylocalauthoritiesgrantsupport(ConseilRe´gionaldeBourgogneGrantnumberPARIAgrale11[http://region-bourgogne.fr/Le-
plan-d-actions-regional-pour-l-innovation-PARI,40,4438]).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparation
ofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction is up-regulated at 37uC [19,20] while expression is repressed at
lower temperatures. Another example is the down-regulation of
ListeriamonocytogenesisaGram+bacteriumcharacterisedbyalow mobility andchemotaxisgenes at 37uC [21].
G+Ccontent.Oneofthestrikingfeaturesofthisbacteriumisthe
In the present paper, we investigated how temperature affects
varietyofitslifestyles[1].Indeed,asasaprophyticbacterium,L.
Agr-dependentregulationthroughacombinationofphysiological
monocytogenes is detected in many habitats such as soil [2,3],
testsanddifferentialtranscriptomeanalysesduringgrowthat25uC
vegetation [4], water systems [5], farm environments [6,7], food-
and37uC.
stuffandthefoodprocessingenvironment[8]wherethebacterium
colonises abiotic surfaces and form biofilms [9]. Another facet of
Results
the ecology of this bacterium is its ability to survive in the
gastrointestinal tract of animals and Humans, to multiply L. monocytogenes Gene Expression Patterns in the
intracellularly and eventually to cause life-threatening diseases
Parental and DagrA Backgrounds are Influenced by
[10,11].
Temperature
The extensive repertoire of transport proteins and regulatory
Figure1.Apresentsthesetsofgeneswithhighertranscriptlevels
genes support the ability to colonise a wide range of ecosystems
at 25uC than at 37uCin L.monocytogenes EGD-eandin the DagrA
[12]. A large percentage of the genome of this ubiquitous
mutant(DG125A).Differentialanalysisoftheleveloftranscriptsof
bacteriumisdedicatedtoregulationincluding209transcriptional
L. monocytogenes EGD-e on the basis of the growth temperature
regulators[12].Amongstthese,AgrAbelongstoanautoinduction
system organised as a four-gene operon agrBDCA [13,14]. AgrC/ showedthat975geneshadhighertranscriptlevelsat25uCthanat
AgrA is a typical two component system that responds to the 37uC (Figure 1.A). Most genes from category 1.5 (Motility and
presence of the auto-inducing peptide. Inactivation of the Agr chemotaxis)and4.3(Phage-relatedgenes)wererepresentedinthis
systemaffectstheabilityofL.monocytogenestoformbiofilmsat25uC groupofgenes(Figure2).Similarly,severalgenesfromcategories
[15]andlowersitsabilitytogenerateinfectioninamurinemodel intermediary metabolism (2.1.1, 2.2, 2.3, 2.5), transport/binding
[16]. proteins (1.2), regulators (3.5.2), ribosomal proteins (3.7.1) and
Environmental adaptation requires integration of a variety of similar to unknown functions (5.2, 6) were most likely to be
environmental cues that results in deep transcriptional reshaping transcribedtohigherlevelsat25uCasindicatedbygeneontology.
duringsaprophytic[17]andinvivo[18]life-styles.Temperatureis Among these, a set of 568 genes was common to EGD-e and
one of these environmental cues that L. monocytogenes integrates in DG125A.
order toregulate gene expression.Expression of virulence factors
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Temperature-DependentAgrRegulation
Figure1.Venndiagramsoftheoverallnumberofgeneswithtemperature-dependentvariationsandrepresentedasspecificor
commontoL.monocytogenesEGD-eandL.monocytogenesDG125A.(A)highertranscriptlevelsat25uC;(B)highertranscriptlevelsat37uC.
doi:10.1371/journal.pone.0043154.g001
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Temperature-DependentAgrRegulation
Figure2.Percentageofgenesvaryingsignificantlyaccordingtothetemperatureanddistributedwithinfunctionalcategories.(red
boxes)L.monocytogenesEGD-e;(blueboxes)L.monocytogenesDG125A.
doi:10.1371/journal.pone.0043154.g002
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Temperature-DependentAgrRegulation
The variation of transcripts of 407 genes was specific to the bindingprotein).TranscriptionofthenadABCoperon,involvedin
EGD-ebackground(Figure1A.).Thelevelsoftranscriptsofthese NADsynthesis,wasalsoincreasedinDG125A.Geneontologydid
genesweresignificantlyhigherat25uC(TableS1).Geneontology not identify significant functional category terms in this set of
identifiedthefunctionalcategories1.1(cellwall),2.2(Metabolism genes.
of amino acids and related molecules), 2.4 (metabolism of lipids) 40genesweredetectedwithlowertranscriptlevelsintheDagrA
and 3.7.2 (Aminoacyl-tRNA synthetases) as significant terms genetic background (Figure 4B, Tables S5, S7). As expected agr
(Figure2).ForexampledltBisinvolvedinD-alanineesterification genes varied with fold changes as high as 36 (agrD). Three genes
of teichoic acid and deletion of the dlt operon affects adhesion to coding for putative secreted proteins (lmo477, lmo478, lmo479)
celllinesandvirulence[22].lmo1076encodesAUTOanautolysin variedby21.4,12.3and4.9respectively.Othergenesincludedin
requiredforvirulence[23,24],proteinsencodedbymreBandmreC this set of genes were lmo0880 (similar to wall associated protein
areinvolvedincell-shapedetermination[25],proteinsencodedby precursor(LPXTGmotif),InternalingenesinlA,inlBandinlHand
tagB,tagD areinvolved inwall teichoicacidsynthesis [26]. thecompleteopuCA-opuCDoperon.Geneontologydidnotidentify
Finally, 108 genes had specifically higher transcript levels at functional category as significant.
25uCthanat37uCintheDagrAmutant(TableS2)butfunctional
category was not a significantterm withinthisset ofgenes. Genes of Transport and Metabolism of Amino Acids and
Resultsofthecomparisonofgeneswithhighertranscriptlevels Related Molecules are Over-expressed in the DagrA
at 37uC are presented Figure 1B. This analysis identified a set of Background during Growth at 37uC
365genesintheparentalstrainEGD-e.Tenfunctionalcategories
The results at 37uC were different. 188 genes had transcript
(1.5, 1.6, 2.1, 2.1.3, 3.3, 3.5.3, 3.5.4, 3.6, 3.7.4, 3.7.5) were not
levels significantly higher in DG125A vs EGD-e (Tables S6, S7).
represented (Figure 2). The transcription of 39 regulators
Geneontologyshowedthatfunctionalcategories1.2–4.3–6.0–1.5–
increased with temperature. Among known regulators, agrA [13],
5.2–2.3–2.2–5.1 weresignificant terms(Figure 5).
zurR [27],codY [28] andprfA [29] varied by 2.2,2.4,3.4 and 4.5
46 genes of the functional category ‘‘Transport/binding
respectively.
proteins and lipoproteins‘‘ (functional category 1.2) had higher
In the DagrA background, transcription of 347 genes increased
transcriptlevelsintheDagrAmutantthaninEGD-ewhengrown
with temperature while a set of 261 genes was common to both
at37uC.Glutamine,aminoacids(L-cyctine,histidine,methionine)
backgrounds. Gene ontology identified functional categories 1.8
spermidine/putrescine and oligopeptides ABC transporters were
(Cell surface proteins), 5.2 (unknown proteins from other
includedinthissetofgenes.Hortologs(lmo2343tolmo2351)ofthe
organisms) and 6.0 (no similarity) as significant terms in this set
ytmIoperonofB.subtilisinvolvedinL-cystinetransport[30]andits
of genes.
transcriptionalregulatorytlI(lmo2352)arerepresentedinthissetof
A set of 104 genes varying significantly with temperature was
genes and most are included in the top 20 of genes with higher
specifictoEGD-e(TableS3).Notably,theleveloftranscriptsof16
transcriptlevels.lmo1740,lmo1739andlmo1738,areco-transcribed
regulatorsincludingAgrAandPrfAwererepresentedinthissetof
[18]andinsilicoanalysissuggesteditwasspecifictohistidine[31].
genes. Finally, a set of 86 genes was specific to DG125A (Table
TheoligopeptideABCtransporters(lmo0135lmo0136lmo0152)are
S4).
structurally related to OppA [32]. lmo0135 encodes a multifunc-
Consideringthecentralroleoftemperatureintheregulationof
tionalproteininvolvedincysteinetransport,acidresistanceandis
virulence,wefocusedonthemajorvirulenceandinternalingenes.
required for virulence [33]. Finally, lmo1516 lmo1517 are co-
Asexpected,thisanalysisshowedthattranscriptionofthecentral
transcribed (operon 251 [18]) and encode an ammonium
virulencegeneclusterwashigherat37uCthanat25uCinEGD-e transporter which is down regulated in PrfA* backgrounds [34].
(Figure 3). However transcription of prfA and plcA did not
Several genes involved in PTS systems (lmo0738, lmo1255,
significantly vary according to the growth temperature in the
lmo1973, lmo1971, lmo1035, lmo1901, lmo2000, bvrB) [35] and
deletion mutant. In the EGD-e background, among internalins,
putative sugar ABC transporters (for example lmo0768, lmo0767
inlB, inlC, inlH, lmo0610, lmo0801, lmo2027, lmo2396 and lmo2821
andlmo0766)hadhighertranscriptlevelsinDG125Acomparedto
had higher transcript levels at 37uC than at 25uC. On the EGD-e.Twogeneswithhighertranscriptlevel(lmo1884andpyrP)
opposite, lmo0327, lmo0514, and lmo2445 were transcribed to
encode permeases for xanthine and uracil. Both genes are co-
higherlevelsat25uCthanat37uC.DeletionofagrAaffectedthese transcribed with genes encoding enzymes that participate to
results. Indeed in DG125A, during incubation at 37uC, higher purineandpyrimidinemetabolism.Indeed,mostgenes(pyrEpyrF
transcription was observed for inlA, inlB, inlH, lmo0331, lmo0333,
pyrD pyrDII pyrAB pyrAa pyrC pyrB pyrP) of the operon encoding
lmo0610, lmo0801, lmo2027 and lmo2821. At 25uC, higher componentsofthepyrimidinebiosynthesispathwayhadtranscript
transcripts were detected for lmo0327, lmo0514, lmo1289, lmo2445
levels higher than 2. Similarly, lmo1885, lmo0132 and purE
and lmo2470.
encodingenzymesofthepurinebiosynthesispathway hadhigher
transcript levels.
Deletion of agrA Results in Moderate Variations of 8outof30‘‘motilityandchemotaxis’’(functionalcategory1.5)
Transcriptome at 25uC genes had higher transcript levels in DG125A. lmo0678 and
Inordertogainabetterunderstandingoftheregulatoryroleof lmo0679codeforproteinssimilartoflagellarbiosyntheticproteins
AgrA, we analysed the differences of levels of transcripts in the FliR and FlhB. Lmo0683 is similar to chemotactic methyltrans-
mutant background compared to the parental background ferase CheR, Lmo0697 is similar to the flagellar hook protein
(DG125A vs EGD-e). At 25uC, this differential analysis showed FlgE, Lmo0699 is similar to the flagellar switch protein FliM,
that 31 genes had higher transcript levels in DG125A in Lmo0711 is similar to flagellar basal-body rod protein FlgC, and
comparison to EGD-e (Figure 4A, Tables S5, S7). The ortholog Lmo0712 is similar to the flagellar hook-basal body complex
of the ytmI operon of B. subtilis encoding an L-cystine ABC protein FliE. We did not evidence any difference in motility
transporter,ariboflavinkinaseandproteinsofunknownfunctions betweenEGD-eandDG125Aandbothstrainswereimmobileat
(lmo2343-lmo2352)belongedtothissetofgenes[30].Othergenes 37uC (datanot shown).
encodingproteinsinvolvedinaminoacidtransportweredetected, Considering ‘‘Metabolism of amino acids and related mole-
for example lmo0152 (similar to oligopeptide ABC transporter- cules’’ (functional category 2.2), 17 genes had higher transcript
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Temperature-DependentAgrRegulation
Figure3.Heatmapofvirulencegenes.
doi:10.1371/journal.pone.0043154.g003
levels including genes encoding enzymes from amino acids Genes Following Similar Trends in the Analysis ‘‘DG125A
biosyntheticpathways,forexampleintheconversionofglutamine vs EGD-e’’ Regardless of Temperature
to glutamate (lmo1733 and lmo1734) and in the arginine and
NineteengeneshadhighertranscriptslevelsinDG125Athanin
aromatic amino acid (lmo1926, hisC, aroB and tyrA) biosynthesis EGD-e regardless of temperature (Figure 4A, Table S7). Five
pathways [36]. genescodeforpeptidesandaminoacidstransporters.Fourgenes
Interestingly, six genes encoding regulators had higher tran- from intermediary metabolism, one monooxygenase and eight
script levels. These included lmo2352 the ortholog of ytlI, the genes codingfor unknownproteins werein thissetof genes.
activator of the ytmI operon encoding an L-cyctine ABC Transcription of 22 genes was lower in the DagrA than in the
transporter [30], codY which encodes a regulator responsive to parentalbackground.Theagroperon,inlA,inlBandgenescoding
GTPandbranchedchainaminoacids[28]andlmo2107codinga forhypothetical proteinswere intheset ofgenes.
putative transcriptional regulator (DeoR family) up regulated Theoverallresultsobservedinthecomparisonsbetweenstrains
during heatshock [37]. (DG125A versus EGD-e at 25uC and 37uC) and between
86 genes had lower transcript levels in the DagrA background temperatures (37uC versus 25uC, backgrounds EGD-e and
(TablesS6,S7).Asexceptedagrgenesvariedwithfoldchangesas DG125A) were confirmed by reverse transcription Real-time
highas29(agrD).Threegenescodingforputativesecretedproteins PCR quantification of the transcripts of a selection of five genes
(lmo477, lmo478, lmo479) varied by 44.6, 41.9 and 43.5 respec- (lm0477, prfA, inlA, lmo1972, lmo2257). As shown figure 6, similar
tively.Fiveregulators(prfA,comK,lmo0083,lmo0602,lmo2744)were trendswereobserved.Furthermore,inordertoconfirmthatthese
representedinthissetofgenes.Over60%ofthegenesofthisset transcriptional differences were a consequence of the deletion of
codeforproteinsofunknownfunctionsandfunctionalcategory6 agrA,wecomplementedDG125Awiththeparentalversionofthe
was a significant term. geneandperformedquantificationoftranscriptsofagrA,lmo0477,
prfA and lmo1972. As presented in supplementary material S1,
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Figure4.VenndiagramsofgeneswithsignificantdifferencesinthecomparisonDG125AversusEGD-eat256Cand376C.(A)genes
withhighertranscriptlevels;(B)geneswithlowertranscriptlevels.
doi:10.1371/journal.pone.0043154.g004
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Temperature-DependentAgrRegulation
Figure5.PercentageofgenesvaryingsignificantlyinthecomparisonDG125AversusEGD-eat256C(blueboxes)and376C(red
boxes),distributedwithinfunctionalcategories.(A)geneswithhighertranscriptlevels;(B)geneswithlowertranscriptlevels.
doi:10.1371/journal.pone.0043154.g005
complementation of DG125A restored the level of transcripts of highertranscriptslevelsinDG125Aand‘‘CodY’’wasasignificant
thisselection of genes. ontologyterm.ThepercentageofvariationwashigherinthesC,
sL,sHandCtsRwith2,4, 25,and7genes respectively.‘‘sH’’
Deletion of agrA Affects Transcription of Sigma Factors, was a significant term in the set of genes with lower transcripts
CodY, CtsR, HrcA, PrfA and VirR Regulons levels.AllofthesegenesarepositivelyregulatedbysH.Regarding
Table 1 shows the percentage of regulons that varied in the thesBregulon,atotalof26genesvaried.Nineofthembelonged
DagrAbackgroundat25uCand37uC.At25uC,thetranscriptionof to the set of genes with higher transcript levels. These genes are
the HrcA and VirR regulons did not significantly vary and a under the negative control of sB except lmo0735, lmo0736,
limited number of genes of the sC, sL, sH, CodY and CtsR lmo0737andlmo0738.Seventeengenesunderthepositivecontrol
regulons had transcripts levels significantly different in DG125A ofsBwereincludedinthesetofgeneswithlowertranscriptlevel.
thaninEGD-ebackground.TheproportionofvariationinthesB Finally,11genespositivelyregulatedbyPrfAhadlowertranscripts
andPrfAregulonswashigher.Thetranscriptionof18genesunder levels in the DagrA than in the parental background at this
thepositivecontrolofthesBwaslower.Similarly,18genesunder temperature.‘‘co-regulationbyPrfAandsB’’,‘‘PrfA’’and‘‘sB’’
the positive control of PrfA were represented in the set of genes wereallsignificanttermsinthissetofgeneswithlowertranscripts
levels.
with lower transcript levels. Interestingly, 14 of them are co-
regulated byboth PrfAandsB(36.8% ofthegenes co-regulated
byPrfAandsB).Geneontologyanalysisidentified‘‘co-regulation Inactivation of AgrA Improves Growth Rates of
byPrfAandsB’’,‘‘PrfA’’,‘‘sB’’assignificanttermsinthesetof Planktonic Cultures but Reduces Salt Tolerance
genes with lower transcripts levels at 25uC. None of the regulon As presented above, deletion of agrA resulted in significant
terms was significant in the set of genes with higher transcript differences of transcription at both temperatures; transcription of
levels at 25uC. genesrelatedtoaminoacidsmetabolismwasgreaterintheDagrA
At 37uC transcription of the HrcA and VirR regulons was not backgroundthanintheparentalstrain,converselytranscriptionof
affected in the mutant background. Twenty genes of the CodY theopuCACDoperonwaslowerwhenAgrAwasnotfunctional.We
regulon, under the negative control of CodY, were detected with therefore investigated whether these variations translated into
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Temperature-DependentAgrRegulation
Figure6.Comparisonoffold-changesdeterminedfrommicroarrayandreversetranscriptaserealtimePCRdata.(%)lmo0477,(e)
prfA,(D)inlA,(O)lmo1972,( )lmo2257;red:DG125AversusEGD-e37uC,green:DG125AversusEGD-e25uC,blue:EGD-e37uCversus25uC,yellow:
DG125A37uCversus25uC.
doi:10.1371/journal.pone.0043154.g006
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InactivationofAgrAEnhancesBiofilmFormationat376C
Table1. Numberofgenes from sigma factors andother
transcriptional regulators regulonswith significant variations Under our experimental conditions, deletion of agrA increased
oftranscripts betweenL. monocytogenes DG125A andL. therateofgrowthat37uC.Wethenassessedwhetherthisgrowth
monocytogenes EGD-e at25uCand 37uC. advantage could be observed under sessile conditions. Indeed, as
shown figure 7, at 37uC L. monocytogenes DG125A produced
significantlyhigheramountsofbiofilmthanL.monocytogenesEGD-
Regulon Percentageofregulon e.Asdescribedbefore[15]deletionofagrAresultedinareduction
of the ability of L. monocytogenes to form biofilms at 25uC. Finally,
256C 376C
both strains produced higheramounts of biofilm at 37uC than at
sB(216)a 8.3*(18)b 12.4*(26) 25uC.
sC(24) 4.2(1) 8.3(2)
Discussion
sL(51) 3.9(2) 7.8(4)
sH(169) 4.1(7) 14.8*(25) Temperature is an important environmental cue that partici-
CodY(86) 2.3(2) 23.2*(20) patestothetransitionofListeriamonocytogenesfromsaprophyticlife
CtsR(64) 3.1(2) 10.9(7) to in vivo adaptation and infection. Consistent with previous
functionalgenomicsstudies[18,38,39,40],underourexperimental
HrcA(61) 0 0
conditions, temperature-dependent transcriptional reshaping was
PrfA(70) 25.7*(18) 15.7*(11)
observed. Transport systems, metabolism, ribosomal proteins,
VirR(17) 0 0 regulators, phage-related functions, motility and chemotaxis was
enhancedatthesaprophytictemperaturewhileat37uC,alimited
*significantontologyterm.
aindicatedinbracketsisthetotalnumberofgenesintheregulon. number of genes were up-regulated including virulence genes.
bindicatedinbracketsisthenumberofgeneswithsignificantvariationof One key finding extracted from the transcriptomic data is the
transcriptlevel. temperature-dependent effect of the inactivation of agrA on
doi:10.1371/journal.pone.0043154.t001
transcription. Indeed, the number of genes with significant
differences of transcript levels was higher at 37uC than at 25uC.
phenotypic traits especially regarding the ability of the mutant to
This suggests that the regulatory role of AgrA is critical at the in
utilizeaminoacidsaswellasitssaltstresstolerance.Wefirstofall
vivo temperature of 37uC. Furthermore, as transcription of agrA
comparedgrowthofbothstrainsat25uCand37uCinthecomplex wassignificantlyhigherat37uCthanat25uC,temperaturemaybe
rich medium TSB and in the chemically defined medium KRM
an important environmental cue for the regulation of the
supplementedwithcasaminoacids.AsshownTable2,at25uCin expression of AgrA. Temperature-dependent expression of the
the chemically defined medium supplemented with amino acids, regulators PrfA [19] and MogR [21] through RNA and protein
thegrowthofstrainDG125AwassignificantlyfasterthanEGD-e thermosensors respectively hasbeen documented.
but the differences were not significant during growth in the Ourdatasuggestthatat37uCAgrAparticipatestothenegative
complex medium TSB. At 37uC, deletion of agrA resulted in a control of the transport of nitrogenous compounds (amino acids,
significantlyfastergrowthunderallconditionstested.WhenNaCl peptides, glutamine, ammonium, spermidine/putrescine), carbo-
wasaddedtoTSB,resultsdiffered.Indeedat25uC,thegrowthof hydrates,xanthineanduracil.Similarly,AgrAmaybeinvolvedin
L. monocytogenes DG125A was affected in TSB supplemented with theregulationofaminoacids,purineandpyrimidinebiosynthetic
1%(w/v),3%(w/v)and5%(w/v)NaCl.At37uC,afteraddition pathways. Indeed, glutamine is an optimal nitrogen source [41].
of NaCl,thedifferenceswere significantwhen3%and5%NaCl Higher transcription of the genes encoding glutamine transport
were added toTSB. proteins and the glutamate synthase was observed. This is the
enzyme that catalyses conversion of glutamine to glutamate.
Glutamate is the major donor of nitrogen to amino acids and
nucleotides[41].Highertranscription ofthe pyroperon encoding
Table2.Growthrates(h21)ofL.monocytogenesEGD-eandL. an uracil permease and the enzymes needed for the synthesis of
uridine-monophosphate (UMP) from glutamine also suggests
monocytogenes DG125A during incubationat25uCand 37uC
higher synthesis of pyrimidines and purines. Deregulation of the
incomplex and definedmedium.
transcription ofalarge numberofproteinsofunknown functions
was alsorecorded.
Highertranscriptionofammonium,aminoacidsandoligopep-
Mediuma 256C 376C tide transporters suggests that nitrogen uptake isenhanced inthe
EGD-e DG125A EGD-e DG125A DagrA background. This was confirmed experimentally as signif-
icantly faster growth was observed in defined medium supple-
TSB 0.31560.025 0.29660.031 0.47660.032* 0.52260.024*
mented with amino acids when AgrA was not functional. These
TSB1%NaCl 0.26660.007* 0.25160.006* 0.45160.019 0.47760.009
deregulations resulted in a growth advantage of the Dagr mutant,
TSB3%NaCl 0.20360.006* 0.17960.003* 0.27260.029* 0.16260.030*
especially during incubationof planktonic andbiofilm culturesat
TSB5%NaCl 0.12160.001* 0.07960.004* 0.24460.013* 0.21160.006* 37uC,butatthesaprophytictemperatureof25uC,deletionofagrA
KRM0.1% 0.13860.003* 0.15960.002* 0.07260.002* 0.08260.002* wasdetrimentaltothegrowthofplanktonicandbiofilmculturesin
casamino TSB andsalt tolerancewas affected at bothtemperatures.
KRM1% 0.13760.004* 0.14960.003* 0.09060.003* 0.10360.003* TwolinesofevidencefurthersuggestthatAgrAmayparticipate
casamino tothetransitionfromsaprophytismtoinvivolifestyles.Firstofall,
transcription of mobility genes and phage-related functions was
*significantdifferences(P,0.05).
aNaClandcasaminoacidswereaddedonaweightpervolumebasis. deregulatedat37uCintheDagrbackgroundwhilethesegenesare
doi:10.1371/journal.pone.0043154.t002 known to be down-regulated at this temperature [18,40].
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Temperature-DependentAgrRegulation
Figure7.Biofilmformationafter16hofincubationat256Cand376C.Thecellsinbiofilmswerestainedwithcristalvioletandmeasured
usingOD590.Thevaluesaremediansofthreeindependentexperimentsincluding5replicatesperexperiment.Barsrepresentstandarddeviations.
Theboxlimitsrepresentthe5thand95thpercentiles.
doi:10.1371/journal.pone.0043154.g007
Conversely,at37uC,transcriptionofgenesencodingthevirulence describedinStaphylococcusaureus[48],auto-inducerpeptidesinthe
factorsInlA,InlBandtheregulatorPrfAwaslowerinthemutant surroundingsofthecellinteractwiththesensorAgrC,ahistidine
background and virulence of Dagr mutants is affected in the kinase that will phosphorylate AgrA. Under this scheme, biotic
murine model [13,16]. information is integrated in the fine-tuning of the physiological
Ascouldbeexpected,ourdatasupportthescenariothattheAgr traits of the cell. It is tempting to speculate that during infection,
system acts in a complex transcriptional regulatory network with throughAgrAregulation,auto-inductionmediatesinformationon
interconnections between regulons. Indeed, 12 regulators were thelisterial bioticstatuswhich,inturn,maymodulateexpression
deregulated to some extent and most of them (10) varied of key factors, including virulence factors, for optimizing in vivo
specifically at 37uC. adaptationandintracellularsurvival.Withinthegutenvironment,
Our data confirm the complex interconnections evidenced in thislisterialbioticstatusmaycombinediffusionsensing[49],asa
the regulatory network of L. monocytogenes. Indeed, the alternative mean to assess proximity to epithelium, and assessment of the
sigma factor sB regulates transcription of mcsA, mscB the number of listerial cell in the vicinity. In this efficiency sensing
modulators of the ctsR operon, hrcA and prfA [42,43,44] while scenario [50], AgrA regulation would optimize the fitness of the
sLregulates transcription of the sigCoperon [42]. listerialcellsbyassessingthebenefitsofproducingcostlyvirulence
Complex overlaps among sB, sC, sH, sL, PrfA, CtsR and factors according tothelocal environment.
HrcA has been evidenced previously [42,45,46,47]. Observation
of overlaps with sB, sH, CodY and PrfA regulons is consistent Materials and Methods
withregulatory redundanciesandsynergisticcontrol oftranscrip-
Strains and Growth Condition
tion of particular sets of genes. Our data suggest a synergistic
activityoftheAgrsystemwithPrfA,sBandsHregulation,and Listeria monocytogenes EGD-e [51] and Listeria monocytogenes
an antagonistic activity with CodY regulation at the in vivo DG125A [15] were kept frozen on glycerol at 270uC. Inocula
temperature. This suggests the existence of an interconnected were prepared by two successive cultures in Tryptone Soy broth
network that regulates transcription as the first stage of the fine- (TSB;BiokarDiagnostics,Pantin,France)inoculatedat1%(vol/
tuning of gene expression in the process of adaptation to ever vol)andincubated16hat25uC.FortranscriptomicFortranscrip-
changingenvironmentalconditions.OurresultssuggestthatAgrA tomicanalyses,culturesweregrowninTSBtoanopticaldensityat
participates to this complex regulatory network, especially in 600nm (OD600) of 0.4 TSB,Brain Heart Infusion(BHI; Biokar
connexion with PrfA, sB, sH and CodY. In this study, the data Diagnostics, Pantin, France) and the chemically defined medium
presented reflects mid-log growth and one can speculate that KRM (Sigma) supplemented with either 0.1% or 1% casamino
expression profiles may change according to the phase of growth acids were used for planktonic growth kinetics and biofilm
and/or environmental conditions. Indeed, AgrA is part of the formation. Experiments were carried out at 25uC and 37uC
auto-inducer peptide responsive Agr auto-induction system. As understatic conditions.
PLOSONE | www.plosone.org 10 September2012 | Volume 7 | Issue 9 | e43154