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High Prevalence of Quorum-Sensing and Quorum-Quenching Activity among Cultivable Bacteria and Metagenomic Sequences in the Mediterranean Sea

Authors:
Andrea Muras
Andrea Muras
Mario López-Pérez at Universidad Miguel Hernández de Elche
Mario López-Pérez
Celia Mayer at Instituto de Investigación Sanitaria de Santiago de Compostela
Celia Mayer
Ana Parga at University of Geneva
Ana Parga
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Abstract and Figures

There is increasing evidence being accumulated regarding the importance ofN-acyl homoserine lactones (AHL)-mediated quorum-sensing (QS) and quorum-quenching (QQ) processes in the marine environment, but in most cases, data has been obtained from specific microhabitats, and subsequently little is known regarding these activities in free-living marine bacteria. The QS and QQ activities among 605 bacterial isolates obtained at 90 and 2000 m depths in the Mediterranean Sea were analyzed. Additionally, putative QS and QQ sequences were searched in metagenomic data obtained at different depths (15-2000 m) at the same sampling site. The number of AHL producers was higher in the 90 m sample (37.66%) than in the 2000 m sample (4.01%). However, the presence of QQ enzymatic activity was 1.63-fold higher in the 2000 m sample. The analysis of putative QQ enzymes in the metagenomes supports the relevance of QQ processes in the deepest samples, found in cultivable bacteria. Despite the unavoidable biases in the cultivation methods and biosensor assays and the possible promiscuous activity of the QQ enzymes retrieved in the metagenomic analysis, the results indicate that AHL-related QS and QQ processes could be common activity in the marine environment.
Cultivable bacteria concentration (colony forming units (CFU)/mL, average ± s.d., n = 5) obtained in the photic (90 m, white bars) and aphotic (2000 m, black bars) samples for the culture media TSA-1%NaCl (TSA-1), Marine Agar (MA), Marine agar diluted 1:100 in seawater (MA 1/100), and Filtered autoclaved sea-water enriched with polymers (FAS-POL).
Cultivable bacteria concentration (colony forming units (CFU)/mL, average ± s.d., n = 5) obtained in the photic (90 m, white bars) and aphotic (2000 m, black bars) samples for the culture media TSA-1%NaCl (TSA-1), Marine Agar (MA), Marine agar diluted 1:100 in seawater (MA 1/100), and Filtered autoclaved sea-water enriched with polymers (FAS-POL).
… 
(A) Bacterial diversity in cultivable bacteria isolated from photic (90 m depth, n = 231) and aphotic (2000 m depth, n = 374) samples; (B) relative abundance of the 13 most abundant genera in 90 and 2000 m depth samples. The genera represented by a single isolate are grouped as "other".
(A) Bacterial diversity in cultivable bacteria isolated from photic (90 m depth, n = 231) and aphotic (2000 m depth, n = 374) samples; (B) relative abundance of the 13 most abundant genera in 90 and 2000 m depth samples. The genera represented by a single isolate are grouped as "other".
… 
(A) Percentage of the isolated strains with the ability to activate the N-acyl homoserine lactone (AHL) sensor Agrobacterium tumefaciens NTL4 in the 90 m (white bars) and 2000 m (black bars) samples; (B) percentage of isolates with quorum-quenching (QQ) activity against C6 and C12-HSL isolated from each culture media in the 90 m (white bars) and 2000 m (black bars) samples, as confirmed by using the Petri dish solid assay with Chromobacterium violaceum CV026 and VIR07. Media used: tryptone soy agar 1% NaCl (TSA-1), marine agar (MA), diluted marine agar (MA 1/100), and filtered autoclaved seawater medium (FAS) supplemented with 0.5 g/L polymers: agarose, chitin and starch (FAS-POL).
(A) Percentage of the isolated strains with the ability to activate the N-acyl homoserine lactone (AHL) sensor Agrobacterium tumefaciens NTL4 in the 90 m (white bars) and 2000 m (black bars) samples; (B) percentage of isolates with quorum-quenching (QQ) activity against C6 and C12-HSL isolated from each culture media in the 90 m (white bars) and 2000 m (black bars) samples, as confirmed by using the Petri dish solid assay with Chromobacterium violaceum CV026 and VIR07. Media used: tryptone soy agar 1% NaCl (TSA-1), marine agar (MA), diluted marine agar (MA 1/100), and filtered autoclaved seawater medium (FAS) supplemented with 0.5 g/L polymers: agarose, chitin and starch (FAS-POL).
… 
(A) Relative frequencies of AHL synthases and AHL receptors; (B) relative frequencies of QQ genes including lactonases and acylases; (C) relative frequencies of AHL-based QS and QQ sequences in comparison to other genes involved in nitrogen (amoC, amt), phosphate (pstA), and sulfur (dsrA, soxB) acquisition, as well as in oxidative metabolism (dmdA). The data was normalized in function of the abundance of the recA and radA housekeeping genes.
(A) Relative frequencies of AHL synthases and AHL receptors; (B) relative frequencies of QQ genes including lactonases and acylases; (C) relative frequencies of AHL-based QS and QQ sequences in comparison to other genes involved in nitrogen (amoC, amt), phosphate (pstA), and sulfur (dsrA, soxB) acquisition, as well as in oxidative metabolism (dmdA). The data was normalized in function of the abundance of the recA and radA housekeeping genes.
… 
Identification and characterization of the marine isolates showing wide Quorum-Quenching (QQ) activity. The presence of QQ activity in the cell extracts and the minimal active concentration of extract (MAC, μg protein/mL cell extract) needed to fully eliminate the activity of 10 μM of C6-HSL was also investigated.
Identification and characterization of the marine isolates showing wide Quorum-Quenching (QQ) activity. The presence of QQ activity in the cell extracts and the minimal active concentration of extract (MAC, μg protein/mL cell extract) needed to fully eliminate the activity of 10 μM of C6-HSL was also investigated.
… 
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Genes2018,9,100;doi:10.3390/genes9020100www.mdpi.com/journal/genes
Article
HighPrevalenceofQuorum‐SensingandQuorum‐
QuenchingActivityamongCultivableBacteriaand
MetagenomicSequencesintheMediterraneanSea
AndreaMuras
1
,MarioLópez‐Pérez
2
,CeliaMayer
1
,AnaParga
1
,JaimeAmaro‐Blanco
1
andAna
Otero
1,
*
1
DepartamentodeMicrobioloxíaeParasitoloxía,FacultadedeBioloxía‐CIBUS,UniversidadedeSantiagode
Compostela,SantiagodeCompostela15782,Spain;andrea.muras@usc.es(A.M.);celiammayer@gmail.com
(C.M.);anapargamartinez@yahoo.es(A.P.);jaimeamaro85@gmail.com(J.A.‐B.)
2
EvolutionaryGenomicsGroup,DivisióndeMicrobiología,UniversidadMiguelHernández,SanJuande
Alicante03202,Spain;mario.lopezp@umh.es
*Correspondence:anamaria.otero@usc.es;Tel.:+34‐881‐816‐913
Received:12December2017;Accepted:12February2018;Published:16February2018
Abstract:ThereisincreasingevidencebeingaccumulatedregardingtheimportanceofN‐acyl
homoserinelactones(AHL)‐mediatedquorum‐sensing(QS)andquorum‐quenching(QQ)
processesinthemarineenvironment,butinmostcases,datahasbeenobtainedfromspecific
microhabitats,andsubsequentlylittleisknownregardingtheseactivitiesinfree‐livingmarine
bacteria.TheQSandQQactivitiesamong605bacterialisolatesobtainedat90and2000mdepthsin
theMediterraneanSeawereanalyzed.Additionally,putativeQSandQQsequencesweresearched
inmetagenomicdataobtainedatdifferentdepths(15–2000m)atthesamesamplingsite.The
numberofAHLproducerswashigherinthe90msample(37.66%)thaninthe2000msample
(4.01%).However,thepresenceofQQenzymaticactivitywas1.63‐foldhigherinthe2000msample.
TheanalysisofputativeQQenzymesinthemetagenomessupportstherelevanceofQQprocesses
inthedeepestsamples,foundincultivablebacteria.Despitetheunavoidablebiasesinthe
cultivationmethodsandbiosensorassaysandthepossiblepromiscuousactivityoftheQQenzymes
retrievedinthemetagenomicanalysis,theresultsindicatethatAHL‐relatedQSandQQprocesses
couldbecommonactivityinthemarineenvironment.
Keywords:quorumsensing;quorumquenching;AHL;lactonase;acylase;marinebacteria
1. Introduction
Quorumsensing(QS)isabacterialcommunicationsystembasedontheproductionandsecretion
ofsmallsignalmoleculescalledautoinducersthataccumulateintheextracellularenvironmentwhen
highcelldensitiesarereached[1].Onceathresholdintracellularconcentrationisachieved,thesignaling
moleculetriggersthesynchronousexpressionofmultiplegenesinthepopulation,initiatinga
coordinatedaction.Althoughdifferenttypesofsignalmoleculeshavebeendescribed[2],thebest
characterizedQSsignalsaretheN‐acylhomoserinelactones(AHLs).TheseQSsignalmoleculesare
constitutedbyahomoserinelactonering(HSL)linkedbyanamidebondtoafattyacid(between4and
18carbons).AHLsareQSsignalsthatareconsideredtypicalofGram‐negativebacteria[3],although
theyarealsoproducedbydifferentcladesofbacteria[4,5],includingtheGram‐positiveExiguobacterium
sp.[6].ThemostcommonAHL‐basedQSsystemcomprisesaLuxI‐typesignalsynthaseandaLuxR‐
typereceptor[7].OtherAHLsynthasesbelongingtothefamiliesLuxM/AinSandHdtShavebeen
described,notsharinghomologywiththeLuxIsynthasefamily[8,9].Somebacteriadonotproduce
AHLsorhavearecognizableLuxIautoinducersynthasebutpossessLuxRhomologs,calledLuxR
Genes2018,9,100 2of20
orphans,thatcaninteractwiththeautoinducerssynthetizedbyotherbacteria[10].Recently,LuxR
homologueshavebeendescribedtoactassensorsforQSsignalsdifferentfromAHLs,makingthe
picturemorecomplex[11].Despitebeingmoreinfrequentlyreported,LuxIorphansarealsopresentin
somebacteria[12].
InspiteofthelowbacterialpopulationintheopenseaandthelowchemicalstabilityofAHLsatthe
highpHofseawater,newevidencereinforcestheideaoftheimportanceofAHL‐mediatedQS
mechanismsinmarineenvironments[3,5,13,14].Morerecently,numerousstudieshavereportedthe
isolationofAHL‐producingbacterialstrainsfrommarinesamples[15–20].Thepresenceofbacteriawith
theabilitytoproduceAHLsinthesemarinemicrohabitatswasreportedinsubtidalbiofilms[21],sponges
[16,22],cnidarians[23,24],andmarinesnow[15,19,25].Itisnowgenerallyacceptedthatthe
AHL‐mediatedQSsystemsplayanimportantroleinrelevantmarineecologyprocessesincludingthe
settlementofinvertebratelarvae[26,27]andofmacroalgaezoospores[28].TheAHLsproducedby
bacteriaassociatedwiththecyanobacteriaTrichodesmiumareproposedtomediateandcoordinatethe
processingandacquisitionofphosphorus[29],alimitingnutrientinoligotrophicopenocean
environments.Furthermore,symbioticandpathogenicinteractionswithaeukaryotichostalsoactas
examplesoftheseecologicallyrelevantniches[28,30].Inaddition,theexpressionofimportantvirulence
genesinmarinefishpathogenicbacteriaiscommonlycontrolledthroughAHL‐mediatedprocesses[31].
ThepresenceofAHLsinopenmarineenvironmentshasbeenalsoreportedusingdirect,
non‐cultivation‐dependentmeasurements[25,32].AlargephylogeneticdiversityoftheAHLs
synthaseswasobservedintheGlobalOceanSampling(GOS)metagenomicdatabase[33],suggesting
thatAHLproductionisawidespreadmechanisminmarineenvironments.TheAHLsareproposed
toparticipateinthemarinecarboncyclebyincreasingtheactivityofcertainkeyhydrolyticenzymes
forthedegradationofparticulateorganiccarboninseawater,playinganimportantroleinthe
remineralisationdepthdistributionofsinkingparticulateorganiccarbon(POC)[25,34].QS‐mediated
processeshavebeensuggestedtobeevenmoreecologicallyrelevantinspecificmarinemicrohabitats
inwhichthebacterialpopulationismoreconcentrated,formingcellclusters[13,14].
SinceQSsystemshaveimportanteffectsintheinteractionsbetweenprokaryotesandalsowith
eukaryotes,itmakessensethatcompetitorshaveevolvedmechanismsforsilencingotherbacterial
QSsystems.Theabilitytodisruptbacterialcommunicationisawidespreadstrategyusedbydifferent
kindsoforganisms:marinealgae[35],terrestrialplants[36],mammaliancells[37],andbacteria
[4,38,39].Thetermquorumquenching(QQ)wascoinedtodescribetheenzymaticinactivationof
AHLQSsignals[40],althoughatpresentthistermisoftenusedinageneralsensetodescribeany
typeofQSdisruption[41].EnzymaticQQisthebeststudiedQSinhibitorystrategy[40].Thegenes
thatcodifythistypeofenzymesareclassifiedintwomaingroups:lactonasesandacylases,although
othertypesofQQenzymeshavealsobeendescribed[39].Thepioneerstudiesontheecological
relevanceofQQprocessescarriedoutwithbacteriaisolatedfromsoilandrhizosphereindicatedthat
2–4.8%ofthesestrainshadtheabilitytointerferewithAHLs[42–44].Morerecentstudiesrevealeda
highprevalenceofQQenzymesinthemarineenvironment:enzymaticQQactivitywasobservedin
bacterialstrainsisolatedfromcorals[23,45],sponges[46],marinebiofilms[47],estuarineandopen
oceansuperficialseawater[48,49],andfishandbivalvehatcheries[50,51],presentinghigher
frequenciesofbacteriawiththiscapability(2–46%)incomparisontoterrestrialsamples[46,48,52–54].
TheimportanceofQQprocessesinthemarineenvironmentwasfurthersupportedbymetagenomic
studiesshowingahighfrequencyofQQenzymesinmarinemetagenomiccollectionsincludingthe
GlobalOceanSamplingcollection[48].DespitetherelevanceofQSandQQinnichemarine
environmentsseemsclearandalltheavailabledatapointstoahighprevalenceofQSandQQ
activitiesintheseawater,thereisnostudyinwhichametagenomicanalysisiscombinedwiththe
analysisoftheQSandQQactivitiesamongcultivableisolatesforthesamesample.Thisdouble
approachwouldallowustoavoidthehandicapsofbothmethodsandtoassesstherelevanceofthese
processesinfree‐livingbacteria.Therefore,theaimofthisworkwastostudytheAHLproduction
anddegradationactivityinfree‐livingbacteriafromtheMediterraneanSeausingtwodifferentbut
complementaryapproaches,suchasfunctionalscreeninginbacteriaabletogrowinstandardculture
Genes2018,9,100 3of20
conditionsandmetagenomicanalysisinordertoimproveourunderstandingoftheecologic
relevanceofAHL‐mediatedprocessesinthemarineenvironment.Themetagenomicanalysiswas
carriedoutfromseawatersamplescollectedfromsixdepthsinthephoticzoneat15mintervals(15,
30,45,60,75,and90m)andfromtwodepthsintheaphoticzone(1000and2000m)atthesametime,
whilecultivablebacteriawereobtainedfromthe90and2000msamplesinordertoassessthespatial
distributionoftheQSandQQprocesses.
2.MaterialsandMethods
2.1.SampleCollection,BacterialQuantification,andStrainIsolation
Eightseawatersamplesfromdifferentdepthswerecollectedformetagenomicanalysesas
describedpreviously[55,56]onOctober15,2015atasinglepointintheMediterraneanSea(37.35361°
N,0.286194°W)bytheresearchvessel‘GarcíadelCid’.Samplesfrom90and2000mdepthwerealso
usedforbacterialisolationandfunctionalscreening.The90msampleisconsideredthelimitofthe
photiczone,withachlorophyll‐a(Chl‐a)valueof0.13mg/m3,atotalorganiccarbon(TOC)of1.35
mgC/L,totalNof6.9μM,totalPof0.25μM,and1.37×105heterotrophicbacteriacounts.The2000
msamplewascharacterizedbyanalmostundetectableChl‐aconcentration(0.01mg/m3),lowerTOC
(0.94mgC/L),highertotalNandp‐values(8.62and0.5μM,respectively),andlowerheterotrophic
bacteriacounts(4.5×104)[55,56].Bothrichandoligotrophicculturemediawereusedforbacterial
isolationaspreviouslydescribed[47].Therichmediaincludedtryptonesoyagar1%NaCl(TSA‐1)
andmarineagar(MA)suitableforeutrophicbacteria,andtheloworganicformulationsincludedMA
diluted1/100withseawater(salinity35g/L)andfilteredautoclavedseawatermedium(FAS)
supplementedwith0.5g/Lofeachofthefollowingpolymers:agarose,chitin,andstarch(FAS‐POL).
Fiveseriesof10‐folddilutionswerepreparedinsterilizednaturalseawaterforeachsampleand
platedintheabove‐mentionedculturemedia.Theplateswereincubatedat22°Cfor15days.Forthe
estimationofcolonyformingunits(CFUs),plateswith30–300colonieswereselected.Atotalof605
strainswererandomlypickedupandisolatedtobeusedforQSandQQfunctionalscreening(Table
S1).AllthemarineisolatesobtainedwereabletogrowonMAat22°C,hencethesecultureconditions
wereselectedasstandardforlaboratorymaintenanceandassays.
2.2.16S‐BasedBacterialIdentification
Theidentificationofthecollectionof605cultivablemarinebacteriawascarriedoutusing200
μLofculturesobtainedinmarinebrothandpooledingroupsof65–80strains.Thesepoolswere
centrifuged,andtheDNAwasextractedwithDNeasyPowerSoilKit(Qiagen®,Hilden,Germany).
Theamplificationof16SrRNAgeneswasperformedusingadiversityassayillumineinhouse
bTEFAP®(Lubbock,TX,USA).PCRwerecarriedoutunderthefollowingstandardconditions:initial
stepof94°Cfor3minfollowedby28cyclesat94°Cfor30s,53°Cfor40s,and72°Cfor1min.
Sequencing(Miseq,Illumina,SanDiego,CA,USA)anddataprocessingwereperformedusing
BLASTnagainstacurateddatabasefromRDPIIandNCBI(MRDNA,Shallowater,TX,USA).
Inordertoanalyzethebacterialdiversityofthecultivablebacteria,the16SrDNAsequences
wereclusteredtooperationaltaxonomicunits(OTUs)definedat95%identityusingCD‐HIT[57].
ThesequenceswereassignedataxonomicidentityusingtheRDPdatabase[58].
Fortheisolatedstrainsshowingwide‐spectrumQQactivity,genomicDNAwasextractedusing
aWizardDNApurificationKit(Promega,Madison,WI,USA),andthebacterial16SrRNAgenewas
amplifiedusingtheuniversalprimers96bfm(5′‐GAGTTTGATYHTGGCTCAG‐3′)and1152uR(5′‐
ACGGHTACCTTGTTACGACTT‐3′)[59].PCRwerecarriedoutunderthefollowingstandard
conditions:initialstepof96°Cfor2minfollowedby35cyclesat95°Cfor1min,53°Cfor30s,and
72°Cfor2min.The16SrRNAsequenceswereidentifiedusingtheweb‐basedtoolEzTaxon[60].
2.3.Quorum‐SensingActivityAssay
Genes2018,9,100 4of20
Themarinebacterialcollection(605strains)wasscreenedforthestrainsʹcapabilitytoactivate
theAHLbiosensorAgrobacteriumtumefaciensNTL4[61,62].Thestrainswereculturedinmicrotiter
platesin200LofMarineBroth(MB)for48h.Theplateswerecentrifuged,andthesupernatants
weretransferredtoanewplate.ThepHofthesupernatantswascheckedtobelessthanpH8inorder
toavoidlactonolysisoftheAHLsproducedathighpHvalues[63].ThepresenceofAHLsafterthe
incubationperiodwasdetectedbyadding50LofamixtureofsoftAgrobacterium(AB)medium
[61](0.2%agar)with5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyroside(X‐GAL,80μg/mL)andan
overnightcultureofA.tumefaciensNTL4(1:5)ontopofthesupernatantsinmicrotiterwells.The
plateswereincubatedfor6–8hat30°C,andtheproductionofbluecolouronthesurfaceofthewells
waschecked.ABmediumpH6.5plustheC6‐HSL(10μM)wasusedascontrol.A.tumefaciensNTL4
wasculturedat22°CinLBorABmediumsupplementedwith30μggentamycin/mL.
Thecapacityofthestrainspresentingwide‐spectrumQQactivitytoactivatethesensorA.
tumefaciensNTL4wasconfirmedwithadisk‐diffusionagarplateassay[modifiedfrom50]atdifferent
times.OnemLofanovernightshakencultureofthebiosensorwasmixedwith4mLofsoftAB
medium(0.8%)withX‐GAL(80μg/mL)tocovertheABagarplates.Oncetheplateshadsolidified,
10μLofsupernatantfromthe24hand48hculturesofthe12wide‐spectrumQQstrainswasloaded
inantibiogramdisksanddepositedontheplates.Theplateswereincubatedat22°Cfor24h.PBS
pH6.5plusC6‐HSLwasusedasacontrol,andthepresenceofablueinductionhaloaroundthedisks
waschecked.ThestockofC6‐HSLwaspreparedinacetonitrileataconcentration1mg/mL.This
stocksolutionwasdilutedinPBStoafinalconcentrationof10μMandaddedtothedisks.
2.4.Quorum‐QuenchingActivityAssay
TheQQactivityofthestrainswastestedusingasolidmicrotiterplateassayscarriedoutwith
theAHLbiosensorsChromobacteriumviolaceumVIR07[64]forC12‐HSLandC.violaceumCV026[65]
forC6‐HSL.Twohundredmicrolitersofthe48hculturescarriedoutinmicrotiterplatesinMBwere
centrifuged,andthepelletswerewashedwithphosphatebufferedsaline(PBS)pH6.5and
resuspendedinanother200μLofthesamebuffer.Thesecellsuspensionswereusedforlive‐cellAHL
degradationassaybyaddingeitherC6‐HSLorC12‐HSL(10μMinPBS,preparedfromaconcentrated
stock—1mg/mLinacetonitrile)andincubatingfor24hat22°C.ThepresenceofAHLsafterthe
incubationperiodwasdetectedbyadding50LofamixtureofsoftLuria–Bertani(LB)(0.2%agar)
andanovernightcultureofthecorrespondingC.violaceumbiosensorontopofthecellsuspensionin
microtiterwells.Theplateswereincubatedfor24hat30°C,andtheproductionofviolaceinwas
observed.PBSpH6.5plusthecorrespondingAHL(10μM)wasusedasacontrol[66].Thesame
assaywasusedtocheckthecapacitytodegradetheoxo‐substitutedAHLsOC6‐HSLandOC12‐HSL.
InordertodetectfalsepositivesderivedfromtheinhibitionofthegrowthoftheC.violaceum
biosensors,allpositivestrainswerere‐isolated,andtheiractivityconfirmedwithaPetridishsolid
bioassayasdescribedpreviously[47].ThePetridishbioassaysallowdistinguishinggrowth
inhibition(presenceofatransparenthaloaroundthewell)fromQSinhibition.IntheC.violaceum
assay,becauseofthehighconcentrationofaddedexogenousAHL(10μM),theabsenceorreduction
oftheviolaceinhalowasconsideredindicativeofenzymaticdegradation.Inthisassay,thepresence
ofaninhibitorysubstanceisgenerallyvisualizedasaclear,nottransparent,haloaroundthewell,
surroundedbyanexternalhaloofviolacein.Nevertheless,thepresenceofaveryhighamountofa
QSinhibitorcounteractingtheactionoftheexogenousAHLcouldnotbefullyexcluded.The
biosensorstrainsweremaintainedinLBplatessupplementedwithkanamycin(30μg/mL).
2.5.CharacterizationofAHL‐DegradationActivity
Crudecellextracts(CCE)wereobtainedaspreviouslyreported[67].Briefly,thecellbiomass
wasobtainedbycentrifugation,resuspendedinPBS,sonicatedonice,andcentrifugedagain.Thecell
extractobtainedwasfilteredthrougha0.20μmfilterandstoredat4°C.Todeterminetheminimal
activeconcentration(MAC)oftheCCEs,C6‐HSL(10μM)wasexposedtodifferentdilutionsofCCEs,
Genes2018,9,100 5of20
inPBSpH6.5.Themixturewasincubatedfor24hat22°C,andthepresenceofasignalwasdetected
usingtheC.violaceumCV026Petridishbioassay.ThecontrolwellswerefilledwithsterilePBSpH
6.5plusAHL(10μM).
2.6.MetagenomicSamples
Eightsamples(Med‐OCT2015‐15m,Med‐OCT2015‐30m,Med‐OCT2015‐45m,Med‐OCT2015‐
60m,Med‐OCT2015‐75m,Med‐OCT2015‐90m,Med‐OCT2015‐1000m,andMed‐OCT2015‐2000m)
[55,56]fromdifferentdepthsweretakenformetagenomicanalysesonOctober15,2015atasingle
samplingsiteintheWesternMediterranean(37.35361°N,0.286194°W).Sixsampleswereobtained
fromtheuppermost100mat15mintervalsusingahoseattachedtoaCTD(Seabird).Anothertwo
samplesfromthedepthsof1000mand2000m,weretakenthenextday(October16)intwocasts
(100Leach)usingtheCTDrosette.
Allseawatersamplesweresequentiallyfilteredonboardthrough20,5,and0.22μmporesize
polycarbonatefilters(Millipore,Billerica,MA,USA).Allfilterswereimmediatelyfrozenondryice
andstoredat−80°Cuntilprocessing.DNAextractionwasperformedfromthe0.22and5μmfilters,
aspreviouslydescribed[68].ThemetagenomesweresequencedusingIlluminaHiseq‐4000(150bp,
paired‐endread)(Macrogen,Seoul,Korea).
2.7.MetagenomicAnalysis
Themetagenomicanalysis(readpre‐processing,assemblyandgenepredictionandannotation)
wascarriedoutaspreviouslydescribed[56].Inbrief,eachmetagenomewasassembled
independentlyusingIDBA‐UD[69].Thegenesobtainedontheassembledcontigswerepredicted
usingProdigal[70]transferRNA(tRNA),andtherRNAgeneswerepredictedusingtRNAscan‐LE
[71],ssu‐align[72],andmeta‐rna[73].Ataxonomicandfunctionalannotationwasperformed
comparingthepredictedproteinsequencesagainstNCBINR,COG[74],andTIGFRAM[75]
databaseswereperformedusingUSEARCH6[76].USEARCH6withane‐value<1e‐5wasalsoused
toidentifypotential16Ssequencesfromasubsetof10millionreadsforeachmetagenomeagainst
RDPdatabase[77].Thesecandidateswerethenalignedtoarchaeal,bacterial,andeukaryal16S/18S
rRNAHMMmodels[78],usingssu‐aligntoidentifytruesequences[72],usingathresholdof
sequenceidentity≥80%andalignmentlength≥90bp.Inordertoanalyzethe16SrDNAdiversity,
identicalsequences(99.9%ofidentity)frombothdatasetswereremovedusingCD‐HITsoftware[57].
Therestofthesequenceswereclusteredtooperationaltaxonomicunits(OTUs)definedat95%
identity.TaxonomicaffiliationsoftheseclusterswereassignedusingtheassignedRibosomal
DatabaseProject(RDP)database[58].
OnlyAHLlactonaseandAHLacylasegeneswithdemonstratedactivitywereusedwithinthe
QQenzymes(Supplementarymaterial).TheanalysisofotherQS‐relatedgenes,suchasthosefor
AHLsynthases(LuxI,AinS,andHdtS)andAHLreceptors(LuxRandAinR)aswellasthegenefor
thesynthaseresponsibleforproducingtheAI‐2signal(LuxS),wascarriedoutbyaligningthe
metagenomicreadsagainsttheNRdatabaseusingDIAMOND[79](blastxoption,tophit,≥50%
identity,≥50%alignmentlength,e‐value<10−5).Theabundanceofthesegeneswasnormalizedbythe
numberofreadsmatchingrecA+radAsequencesforeachmetagenome.Thereadsthatgavehitto
viraloreukaryalproteinswerenottakenintoaccount.Inordertoanalysetherelativeabundanceof
theQQenzymesinthewatercolumn,weappliedthesamesequencesearchforgenesinvolvedinthe
normalmetabolismderivedfrommarinebacteria,suchasnitrogen(amoC,amt),phosphate,(pstA),
sulfur(dsrA,soxB),andgeneraloxidativemetabolism(dmdA)[48,80].
2.8.DataAvailability
ThemetagenomicdatasetsusedinthisstudyweresubmittedtoNCBISRAandareavailable
underBioProjectsaccessionnumberPRJNA352798(Med‐OCT2015‐15m,Med‐OCT2015‐30m,
Genes2018,9,100 6of20
Med‐OCT2015‐45m,Med‐OCT2015‐60m,Med‐OCT2015‐75m,Med‐OCT2015‐90m,Med‐OCT2015‐
1000mandMedOCT2015‐2000m).
2.9.StatisticalAnalysis
TheeffectsofdepthandculturemediaonthenumberofCFUs/mLwasanalysedwiththe
non‐parametricMann–Whitneytestatsignificancelevelp<0.05,withIBMSPSSstatisticsV20program.
3.Results
3.1.BacterialGrowthandIsolation
ThenumberofCFUs/mLwassignificantlyhigherinthe2000m(0.8–5.8×103CFUs/mL)thanin
the90mwatersample(0.2–0.6×103CFU/mL)forthedifferentculturemediatested(Mann–Whitney
Test,p<0.05),despitetheisolationconditionsexcludedtheretrievalofbarophilicorpsychrophilic
strainsfromthedeep‐seasample.TheculturemediadidnotaffectthenumberofCFUs/mLobtained
inthe90m,photicsample(Figure1,Mann–WhitneyTest,p>0.05).Onthecontrary,theCFUs
obtainedinMAandFAS‐POLculturemediaweresignificantlyhigherinthesamplefrom2000m,
yieldingthreetimesmoreCFUsthantheothermedia(Figure1,MannWhitneyTest,p<0.05).
Figure1.Cultivablebacteriaconcentration(colonyformingunits(CFU)/mL,average±s.d.,n=5)
obtainedinthephotic(90m,whitebars)andaphotic(2000m,blackbars)samplesfortheculture
mediaTSA‐1%NaCl(TSA‐1),MarineAgar(MA),Marineagardiluted1:100inseawater(MA1/100),
andFilteredautoclavedsea‐waterenrichedwithpolymers(FAS‐POL).
3.2.TaxonomicDiversityoftheCultivableStrains
Atotalof605isolates,231fromthe90mand374fromthe2000msample,werecollectedand
screenedfortheircapacitytoactivateaQSbiosensorandfortheirQQactivityagainstAHLs
(SupplementaryTableS1).ThemostcultivablestrainsbelongedtoGammaproteobacteria(34.88%),
Firmicutes(30.95%),andAlphaproteobacteria(17.44%).MembersoftheActinobacteria(6.84%)and
Bacteroidetes(7.14%)werelessrepresented(Figure2A).Firmicuteswerehighlyrepresentedinthe
cultivablecollection(30%inbothsamples),incomparisonwiththedataobtainedfromthe
metagenomicanalysisatthesamedepth(<1%)[56].Gammaproteobacteria(35%)werealso
overrepresentedinthe90msampleincomparisonwiththemetagenomicdata(13.56%).Therelative
abundanceatthefamilylevelshowedsimilarprofilesatbothdepths,exceptforVibrionaceaeand
Rhodobacteraceaewhichwereonlyidentifiedat2000m(Figure2A).However,whentheOTUswere
clusteredat95%ofidentityfrombothdatasetstoseparatethematgenuslevel,56and82generacould
beidentifiedat90and2000m,respectively,showingthattherewasagreatergeneticdiversityat2000
mthatcouldnotbeappreciatedathighertaxomoniclevels.Inthefuture,thegenomesequencingof
theseorganismscouldclarifyandshedlightonmanyofthedifferencesbetweenthesetwodepths.
Despiteofthis,thetwocollectionsofcultivablebacteriashared11ofthe13mostabundantgenera
Genes2018,9,100 7of20
(Figure2B).Surprisingly,Bacilluswasthemostabundantgroupamongthecultivablebacteria,
representing15.74%and16.6%,inthe90and2000msamples,respectively.ThegeneraMicrobacterium
(2.38%)andSphingomonas(2.38%)wereexclusivelyidentifiedinthesamplefromthe90mdepth,
meanwhilethegenusPantoeaandVibrioappearedonlyinthesamplefrom2000m(Figure2B).
Figure2.(A)Bacterialdiversityincultivablebacteriaisolatedfromphotic(90mdepth,n=231)and
aphotic(2000mdepth,n=374)samples;(B)relativeabundanceofthe13mostabundantgenerain90
and2000mdepthsamples.Thegenerarepresentedbyasingleisolatearegroupedas“other”.
3.3.Quorum‐SensingandQuorum‐QuenchingActivitiesamongCultivableBacteria
Theactivationofthebeta‐galactosidasegeneofthesensor,whichsuggeststhepresenceofAHLs
intheculturemedia,wasrelativelyfrequentamongthe605marineisolates(20.84%).Thenumberof
positiveswashigherinthesamplesfrom90m(37.66%)thaninonesfrom2000m(4.01%)(Figure
3A).TheoligotrophicMA1/100culturemediumallowedtheisolationofthehigherpercentageof
strainswithputativeQSactivityatboth90(60%)and(12.67%)2000m.Onthecontrary,thestrains
isolatedfromTSA‐Ipresentedalowercapacitytoactivatethereporter(4.76and2.52%for90and
2000msamplesrespectively).
Genes2018,9,100 8of20
Figure3.(A)PercentageoftheisolatedstrainswiththeabilitytoactivatetheN‐acylhomoserine
lactone(AHL)sensorAgrobacteriumtumefaciensNTL4inthe90m(whitebars)and2000m(blackbars)
samples;(B)percentageofisolateswithquorum‐quenching(QQ)activityagainstC6andC12‐HSL
isolatedfromeachculturemediainthe90m(whitebars)and2000m(blackbars)samples,as
confirmedbyusingthePetridishsolidassaywithChromobacteriumviolaceumCV026andVIR07.
Mediaused:tryptonesoyagar1%NaCl(TSA‐1),marineagar(MA),dilutedmarineagar(MA1/100),
andfilteredautoclavedseawatermedium(FAS)supplementedwith0.5g/Lpolymers:agarose,chitin
andstarch(FAS‐POL).
Thecapacityofthemarinebacterialcollectiontointerferewithbothlong‐ (C12‐HSL)and
short‐chain(C6‐HSL)AHLswasfirsttestedusingaC.violaceum‐basedbioassayin96‐wellmicrotiter
plates.NoneoftheQQpositivesinthepreliminaryassayshowedinterferencewiththegrowthofthe
biosensors.TheabilitytoquenchtheQSsignalC12‐HSLwasobservedinalargenumberofcultivable
bacteria(38.24%).Theaverageactivitywashigherinthe2000m,aphoticsample(47.05%)thaninthe
90mphoticone(29.43%,Figure3B).Amongthe68strainsisolatedfromthe90mdepthwiththe
capacitytoquenchC12‐HSL,fourstrains,representing1.73%ofthetotalstrains,werealsoableto
eliminatetheC6‐HSLQSsignal,andasimilarpercentagewasobtainedfortheisolatesfromthe2000
msample(2.12%).AlltheisolateswiththeabilitytointerferewithC6‐HSLcouldalsodegrade
C12‐HSL,butnoisolatewasfoundthatcouldonlyeliminatetheactivityoftheshort‐chainAHL.The
oligotrophicMA1/100culturemediumallowedtheisolationofthehighestpercentageofstrainswith
QQactivityagainstC12‐HSL(84.5%)inthe2000msample.Onthecontrary,inthe90msample,the
richculturemedia(TSA‐IandMA)presentedthehighestpercentagesofQQactivity(50–52%).The
mosteffectiveculturemediumtoisolatestrainswithQQactivityagainsttheshort‐chainsignal
Genes2018,9,100 9of20
C6‐HSLweretheoligotrophicFAS‐POL(3.22%)andMA1/100(9.85%)forthesamplesfrom90and
2000m,respectively.
3.4.IdentificationoftheWide‐SpectrumQuorum‐QuenchingStrainsandCharacterizationofQuorum‐
QuenchingActivity
ThetaxonomiclabelofthebesthitisshowninTable1forthe12strainspresenting
wide‐spectrumQQactivity.Thefourmarinestrainsselectedfromthe90msamplebelongedto
Firmicutes(Planomicrobiumchinense),Alphaproteobacteria(Sphingopyxisalaskensis,Erythrobacter
citreus),andActinobacteria(Microbacteriumschleiferi).Amongtheeightstrainsfromthedeepest
sample,oneofthembelongedtoBetaproteobacteria(Ralstoniapickettii),sixtoAlphaproteobacteria(5
Erythrobacterflavusstrains,Citomicrobiumsp.),andonetoGammaproteobacteria(Pantoeasp.).Despite
thehighnumberofBacillussp.strainspresentinthecollections,noBacillusstrainwithwide‐spectrum
QQactivitywasidentified.
Table1.IdentificationandcharacterizationofthemarineisolatesshowingwideQuorum‐Quenching
(QQ)activity.ThepresenceofQQactivityinthecellextractsandtheminimalactiveconcentrationof
extract(MAC,μgprotein/mLcellextract)neededtofullyeliminatetheactivityof10μMofC6‐HSL
wasalsoinvestigated.
LiveCell CellExtrac
t
MACC6‐
HSL(μg
Protein/mL
CellExtract)
StrainClosestCultivated
Bacteria
%IDat16S
rRNAGene
Locus
C6‐
HSL
OC6‐
HSL
C12‐
HSL
OC1
2‐
HSL
C6‐
HSL
C12‐
HSL
90m
1F1Planomicrobiumchinense99.93 + + + + ++104.7
2E12Sphingopyxisalaskensis99.93 + + + + ++18.67
2G12Erythrobactercitreus99.04 + + + + ++1918
3A3
M
icrobacteriumschleiferi99.71 + + + + ‐ +nd
2000m
2F1Ralstoniapickettii95.79 + + ± + ++17.6
3G7Erythrobacterflavus99.34 + + + + ++786
4B4Erythrobacterflavus99.34 + + + + ++216
4B7Pantoeasp.96.01 +‐+‐‐‐ nd
4B9Erythrobacterflavus99.71 + + + + ++961
4B10Erythrobacterflavus99.49 + + + + ++223.9
4B12Erythrobacterflavus99.49 + + + + ++965
4C3Citromicrobiumsp.98.33 + + + + ‐ +nd
Nd:Notdetermined.
Theisolate2F1mayrepresentanewspecieswithinthegenusRalstoniabecauseofthelow
identitywiththeknownspeciesofthegenus(95.79%).Thecellextractsofthreeisolates,3A3
(Microbacteriumschleiferi),4B7(Pantoeasp.),and4C3(Citromicrobiumsp.)didnotpresentQQactivity
againstC6‐HSL.Theabilitytointerferewithoxo‐substitutedAHLswasalsotestedforthe12selected
strains(Table1).Mostofthemarinestrainswereabletoinactivatetheoxo‐substitutedAHL,except
forstrain4B7(Pantoeasp.)thatdidnotshowQQactivityagainstanyofthesubstitutedAHLstested,
namelyOC6andOC12‐HSL.Inordertochecktherelativeactivityofthestrains,theminimalactive
concentration(MAC,μgprotein/mL)wascalculatedinthecellextracts.Themarineisolateswiththe
highestactivitywere2E12(Sphingopyxisalaskensis)and2F1(Ralstoniasp.),sincetheirQQactivitywas
atleastoneorderofmagnitudehigherthanthatoftheotherQQstrains(<20μg/mL).
Amongthe12strains,Sphingopyxisalaskensis2E12andErythrobactercitreus2G12couldactivate
thebiosensorA.tumefaciensNTL4using24hsupernatants.Thisactivitywasmaintainedin48h
supernatantsonlyforSphingopyxisalaskensis2G12,butdisappearedinErythrobactercitreus2G12.
AHL‐likeactivitywasalsodetectedin48hsupernatantsofCitromicrobiumsp.4C3.
3.5.Quorum‐SensingandQuorum‐QuenchingGenesinMetagenomicSamples
AsearchforQS‐relatedgenes,AHLsynthases(LuxI,AinSandHdtS)andAHLreceptors(LuxR
andAinR)aswellasthesynthaseresponsibletoproducetheAI‐2signal(LuxS),wascarriedoutin
Genes2018,9,100 10of20
themetagenomicsamplesfromthedepthprofile.TherelativeabundanceofQS‐relatedgenesseemed
toslightlydecreasewithdepthinthefirst100m(7.59–3.41,Figure4A).However,ahighrelative
abundanceofAHLreceptorswasobservedinthetwoaphoticzonesamples,the1000m(9.02)and
especiallythe2000msamples(20.52).Remarkably,anextremelylowpresenceofAHLsynthaseswas
foundinthemetagenomicanalysisalongthewholewatercolumnincomparisonwiththereceptors
frequency(Figure4A).Surprisingly,noAI‐2synthaseLuxShomologsweredetectedinanyofthe
testedsamples,despitethepresenceofVibriointhebacterialcultivablecollection(3.5%).
Figure4.(A)RelativefrequenciesofAHLsynthasesandAHLreceptors;(B)relativefrequenciesof
QQgenesincludinglactonasesandacylases;(C)relativefrequenciesofAHL‐basedQSandQQ
sequencesincomparisontoothergenesinvolvedinnitrogen(amoC,amt),phosphate(pstA),andsulfur
(dsrA,soxB)acquisition,aswellasinoxidativemetabolism(dmdA).Thedatawasnormalizedin
functionoftheabundanceoftherecAandradAhousekeepinggenes.
WesearchedforthepresenceofAHL‐lactonasesandacylasesgenes,usingsequenceswith
provedactivity,inthesamemetagenomicsamples.AmongtheQQgenes,AHLacylasesweremore
frequentthanAHLlactonasesinmostmetagenomicsamples(0.99,Figure4B).Theanalysisshowed
aclearincreaseofthefrequencyofacylasesat2000m(0.99,Figure4C),inthesamesamplethat
yieldedthehighestnumberofLuxRhomologues(Figure4A).TheincreaseintotalQQsequencesat
2000mwasduetoanincreaseinacylasesequences,sincetherelativeabundanceofacylasesequences
wassimilarinthefirst1000m(0.11–0.32)butincreasedsharplyat2000m(0.99)(Figure4B).In
contrast,thelactonasesequencesreachedthehighestpresenceinthe75msample(0.22)and
decreasedinthesamplesfromgreaterdepths.
InordertoanalyzetherelativeabundanceoftheQQenzymesinthewatercolumn,weapplied
thesamesequencesearchforgenesinvolvedinthenormalmetabolismofmarinebacteria,suchas
thoseinvolvedinnitrogen(amoC,amt),phosphate,(pstA),sulfur(dsrA,soxB),andgeneraloxidative
metabolism(dmdA)(Figure4C)[43,65].TherelativefrequencyofQS‐ andQQ‐relatedgeneswas
Genes2018,9,100 11of20
smallerthanthefrequencyofthegenesrelatedtothecommonuptaketransportersforammonia(amt)
orphosphate(pstA).However,thefrequencywasinthesamerangeofthoseofamoCandofgenes
relatedtosulfurmetabolism(dsrA,soxB).
4.Discussion
Understandinghowbacteriainteractwitheachotherisessentialforpredictingtheirroleinthe
marinemicrobialenvironmentandtheirpotentialimpactsonmarineecology[14].Theabilityof
microorganismstomodulatethebehaviorofanentirepopulationthroughthecoordinationand
regulationofgeneexpressionisviewedasanevolutionaryadaptationtosurviveinachanging
environment[81].DespitetheQSandQQinhibitionprocesseswerediscoveredinthemarine
environment[35,82],littleattentionhasbeenpaidtotheecologicalsignificanceofQSandQQ
mechanismsinseawater,andthisissuehasbeenthecenterofcontroversy[13,14].Increasing
evidencepointstoanimportantroleofAHL‐mediatedQSindifferentmarineenvironments[14],and
thefrequentpresenceofQQactivityamongmarinebacteriaisolatedfromseveralnichehabitats
furthersupportstheecologicalsignificanceofAHL‐dependentQSinthehighlycompetitivemarine
environment.HighQQactivitymaybeinterpretedastheresultofeitheranadaptativetraitofstrains
presentingthisQQcapacityinasituationinwhichQSprocessesincreasebacterialspeciesfitness,or
asituationinwhichtheconcentrationofAHLmoleculesishighenoughtoconstituteasignificant
sourceofcarbon.Inpreviousworks,ahighnumberofstrainswithQQactivity(2–46%)wereisolated
fromdifferentmarineenvironments[46–48,50–54].However,themajorityofthestudiesonlyused
cultivation‐dependenttechniques,introducinganimportantbiasintheevaluationofthesignificance
ofthisactivity.Inthepresentcase,wehavecomparedthepotentialAHLproductionanddegradation
activityinbacteriaisolatedfromtheMediterraneanSeaatdifferentdepthsthroughbiosensor‐based
screeningmethods,withthenumberofputativeQSandQQgenesfoundinmetagenomesobtained
fromthesamemarinesamples.Thissimultaneousstudyallowedustoobtainamorecomplete
assessmentofthepossibleprevalenceoftheseprocesses,avoidingthelimitationsofthe
culture‐derivedandmetagenomicsearchmethodologies.Whileacultivablebacteriaanalysisonly
allowstoevaluateasmallpercentageofthetotalpopulation,metagenomicanalysisresultsdonot
takeintoaccountthepossiblepromiscuityofsubstratesintheretrievedQQsequencesandtheactual
expressionofthesegenesunderenvironmentalconditions.
Ahighercultivablebacterialdensitywasobservedintheaphotic,2000mdepthsample(upto
5.64×103CFU/mL)thaninthe90m,photicsample(upto0.6×103CFU/mL),despitenospecific
culturestrategywasappliedtoretrievepsychrophilicorbarophilicstrains.Theculturemediumused
fortheestimationisanimportantfactor,asdemonstratedbytheresultsobtainedinthe2000mdepth
sample(Figure1)andinpreviousstudiesintheAtlanticSea[42,48].Theoppositetrendwasobserved
inthecountsofheterotrophicbacteriaobtainedbyflowcytometrythatyieldedthreetimesmore
bacteriaat90thanat2000mdepth[56].Thisdiscrepancymayreflectahigherdensityofcultivable,
copiotrophicbacteriaintheaphoticsample,whichisfurthersupportedbythehighercountsobtained
incarbon‐richAMandFAS‐POLculturemediaforthissample.HighertotalPandNwereavailable
at2000m(0.5μMand8.6μMrespectively)incomparisontothesamplefrom90m(0.25μMand6.9
μM),whichmayalsoexplainthehigherCFUs/mLretrievedintheaphoticzonesample.Onthe
contrary,lowerTOCwasavailableinthe2000maphoticsample(0.94mgC/Lagainst1.35mgC/L),
whichcouldgenerateahighlycompetitive,nutrient‐limitedenvironmentintheaphoticsample.
Whileimportantdifferencesinthecommunitystructurewerefoundassociatedwithdepthin
themetagenomicsanalysis[56],thediversityoftheisolatedstrainswasverysimilarinthesamples
collectedat90and2000m,withmoststrainsbelongingtotheProteobacteriaandFirmicutesphyla.
Thisdifferenceisnotunexpected,asonlyabout1%ofthetotalmarinemicrobescanbeisolatedon
artificialmedia,whilethevastmajorityremainsuncultivable[83].Moreover,metagenomesavoidthe
biasimposedbyculturingtechniques,whichprioritizesthegrowthofr‐strategiesmicrobesthatare
expectedtodominatethecultivablepopulation.Proteobacteriawerethemostprevalentlineagein
Genes2018,9,100 12of20
boththemetagenomicdataset(43%)andthecultivablebacteriacollection(52.38%),butthe
percentageofreadingsassociatedwithFirmicuteswaslessthan1%inanyofthedepthsinthe
metagenomicdiversity,whilethisclassrepresentedaround30%ofthecultivablebacteria.
Furthermore,whiletheAlphaproteobacteriadominatethebacterialpopulationintheMediterranean
Sea,decreasinginabundancewithdepth[56,84],inthecultivablecollectionsthisgrouprepresented
only18%withnodifferencebetweenthephoticandaphoticsamples.
ThedataobtainedfromtheMediterraneanSeabacterialcollectionsobtainedfrom90and2000
mdepthsshowedahighpercentageofstrainswiththeabilitytoactivatetheAHL‐reporterA.
tumefaciensNTL4.AlthoughthecultureconditionscouldstronglyaffectQSsignalproduction,and
thedetectionofAHL‐likeactivityintheculturesdoesnotguaranteethatthesignalsareproduced
underenvironmentalconditions[5],theresultssuggestthatAHL‐mediatedQSsystemscouldbea
commoncoordinationmechanisminmarinebacterialcommunities.Itshouldbenotedthatthe
microtiter‐basedscreeningmethoddoesnotallowhighgrowthrates,and,therefore,thenumberof
AHLproducerscouldbeunderestimated,atleastunderlaboratoryconditions,asdemonstratedfor
theidentificationofadditionalpositivestrainswhenculturedinhigher‐volume,shakencultures.A
higherabundanceofputativeAHLproducerswasfoundinthephotic,90msample(37.66%)in
comparisonwiththedeep‐seasample(4.01%).LowerPavailabilityandhigherbacterialdensitymay
justifythehigherrelevanceofQSmechanismsat90m,sinceAHLshavebeenproposedtomediate
andcoordinatethemechanismsofprocessingandacquisitionofP[29].AprevalenceofQSsystems
hasbeendemonstratedinnutrient‐deficientconditions[85],whichcouldalsobecorrelatedwiththe
higherputativeQSactivityfoundunderlaboratoryconditionsamongisolatesobtainedwiththe
oligotrophicculturemediaMA1/100andFAS‐POL.
ThescreeningforQQactivityshowedthat38.24%ofthestrainswereabletointerceptthe
long‐chainQSsignalC12‐HSL.AlthoughAHL‐likeactivitywasalsodetectedinthesupernatants
fromsomeoftheseQQstrains,itisunlikelythattheobservedinhibitoryeffectcansimplybeascribed
totheinhibitoryeffectofnon‐cognateAHLs,sincetheQQactivityisstillpresentinthecellextracts,
andtheconcentrationofthesignalwouldbetoolowtocounteractthehighAHLconcentrationused
intheQQscreeningassays(10μM).Inthesameway,andalthoughwecannotfullydisregardthe
factthatsomeofthestrainswithQQactivitywereproducingahighconcentrationofaQSinhibitory
substance,thehighAHLconcentrationusedinthebioassayssupportsthefactthattheobservedQQ
activityshouldbemainlyofenzymaticnature.Additionalbiochemicalanalysisshouldbeperformed
toconfirmthishypothesis[86].Thehighpercentageofcultivablestrainsidentifiedinbothphoticand
aphoticsampleswiththiscapacityindicatesthatQQmaybeacommonactivityintheMediterranean
Sea,whichisgenerallycharacterizedbyoligotrophy[87].OppositetotheputativeQSactivity,QQ
activityagainstC12‐HSLwasalmost1.63‐foldat2000m(47.05%)incomparisonwiththesample
from90m(29.43%).ThisabundancecorrelateswithlowerPOCavailabilityinthedeepersampleand
maythereforeindicateahighlycompetitiveenvironmentinwhichthebacterialpopulationcapable
ofusingthelong‐chainAHLasasourceofcarbonmayhaveanecologicaladvantage.AlthoughpH,
salinity,pressure,andintensityoftheUVirradiationoftheseawaterhaveasignificantinfluenceon
theproduction,stability,andperceptionoftheQSsignalmolecules[13,24,88,89],thedifferencesin
thephysicochemicalparametersbetweenthetwosamples[56]donotjustifythedifferencesinQS
andQQactivity.
Alargedifferencewasobservedbetweentheabilityofthestrainstodegradethelong‐chain
signalC12‐HSL(38.24%)ortheshort‐chainsignalC6‐HSL(1.93%).Unlikethedegradationof
C12‐HSL,thepercentageofstrainsbeingabletodegradeC6‐HSLwassimilarinbothsamples(1.73
and2.12%).Thislowprevalenceofstrainswithwide‐spectrumQQactivityissimilartothe
percentagereportedforsoilsamplesagainsttheC6‐HSL(2.5%)[43].Themorediffuseddegradation
oflong‐chainAHLssignalsamongmarinebacteriacouldberelatedtotheself‐degradationprocess
thatthelactoneringsuffersatthehighpHofseawater,whichoccursfasterfortheshort‐chainAHLs
[53];therefore,specificenzymaticdegradationwouldbemorenecessaryforthelong‐chain
molecules.ThisdatawouldalsosupporttheideathatQSsignalscanbeusedasanadditionalcarbon
Genes2018,9,100 13of20
andenergysourceundercarbon‐limitationconditions,sincethedegradationofalong‐chainsignal
suchasC12‐HSLcouldcontributemoreefficientlythanthatoftheshorteronestothemetabolic
budgetofthecells.
Thewide‐spectrumQQstrainsidentifiedinthisstudybelongtoAlphaproteobacteria(eight
strains),Betaproteobacteria(onestrain),Gammaproteobacteria(onestrain),Actinobacteria(one
strain),andFirmicutes(onestrain).Additionally,anovelspeciesbelongingtothegenusRalstonia
wasidentified(strain2F1).MicrobacteriumschleiferiandRalstoniasp.,isolatedfrom90mand2000m
depth,respectively,belongtoagenuswithpreviouslyreportedQQactivity[89–92].Onthecontrary,
thisisthefirstreportontheabilitytointerceptAHL‐mediatedcommunicationofmembersfromthe
genusPlanomicrobium,Sphingopyxis,Erythrobacter,Pantoea,andCitromicrobium.Aspreviously
reported[46–48,50],thetaxonomicdiversityfoundinthemarinebacteriawithwide‐spectrumQQ
activityismuchhigherthaninsoilandplantsamples,wheremainlyBacillusstrainswereidentified
[43,44].Surprisingly,inourcase,despitethehighnumberofBacillusstrainspresentinthecollection
(15%),noneofthemshowedwide‐spectrumQQactivity.MostofthestrainswithQQactivityisolated
inthisstudybelongtotheAlphaproteobacteria,includingrepresentativesofthegenus
Citromicrobium,Erythobacter,andSphyngopyxis.Alphaproteobacteriarepresentmorethan15%ofthe
cultivablebacteriaanalyzedand25%ofthemetagenomicdiversity(Figure2)[56].Althoughthe
abundanceofErythrobacterstrainsinthecultivablecollectionwassimilarinbothsamples(2.30%),
surprisingly,thosefrom2000mdepthpresentedQQactivitymorefrequently(fivestrainsagainst
one).DifferencesintheQQactivityamongspeciesofthesamegenushavebeenalreadyreported
[66],and,inthiscase,thehigheractivityamongisolatesfromthedeepersamplemayfurthersupport
anadaptativevalueofthisactivityincarbon‐limitedenvironments.Regardingtheactivitypresentin
thecrudecellextract(CCE)ofthewide‐spectrumQQstrains,strains2E12(Sphingopyxisalaskensis)
and2F1(Ralstoniasp.)presentedthestrongestQQactivity,sincetheirMACwere18.67and17.6μg
CCEprotein/mL,respectively.ThisvalueissimilartotheMACreportedforTenacibaculumsp.20J,a
bacteriumwithhighQQactivity[66,67].FurthercharacterizationoftheQQactivityofthesestrains
inordertoconfirmtheirQQenzymaticactivitywithadditionalbiochemicalanalyses[86,93]will
certainlycontributetothesearchfornovelanti‐pathogeniccompoundswithpotentialapplications
tocontrolbacterialinfectionsinplants[42],aquaculture[31,51,67],andmorerecentlyinbiomedicine
[94].
WehaveobservedaclearincreaseinthepresenceoftheQSandQQputativesequenceswith
thesampledepth.Therelevanceofthesegenesisespeciallyevidentintheaphoticsamples,inwhich
theabundanceoftheQSandQQsequenceswashigherthanthatofsequencesrelatedtosulfur(dsrA
andsoxB),nitrogen(amoC),andoxidativemetabolism(dmdA).ThesimilarfrequencyofQQenzymes
inrelationwithotherimportantprocessesmightsuggestapotentialrelevanceoftheseprocessesin
thesea,mainlyindeeperwaters.AfurtheranalysiswouldbenecessaryinordertoassesifQSand
QQgenesareactuallyexpressedinthemarineenvironment.
WhilethemetagenomicdataareconsistentwiththehigherQQactivityfoundamongcultivable
bacteriaindeepersamples,animportantdiscrepancywasfoundbetweenthehigherpresenceof
QS‐relatedgenesinthe2000msampleandthelowerputativeQSactivityinthecultivablebacteria
obtainedfromthesamesample.Asexplainedbefore,thebiasderivedfromtheuseofculture
conditionsnotresemblingthenaturalmarineenvironmentmaybethecauseofthisdiscrepancy.A
recentstudyonfourdifferentEscherichiacolistrainsshowedthatthe47%ofthevarianceinexpression
levelsisdependentprimarilyontheenvironment(medium‐dependentgenes)[95].Therefore,the
presentinvitroexperimentsindicatetheabilityofahighpercentageofthemarinestrainstoactivate
AHLsensorsandquenchtheAHLmolecules;however,furtherenvironmentalinvivostudiesare
neededtoconfirmandcompleteourknowledgeregardingtheroleandrelevanceofAHL‐mediated
QSinthemarineenvironment.
ThehigherpercentageofcultivablestrainswithAHL‐typeQSandQQactivitystronglysupports
thesignificanceofthisactivityinmarinesamples,however,theresultsderivedfromthe
metagenomicsanalysisshouldbeinterpretedwithcaution.First,thedetectionofsequenceswithhigh
Genes2018,9,100 14of20
similaritiesdoesnotensuretheconservationofthefunctionalityortheexpressionofthegeneunder
specificenvironmentalconditions.Secondly,sinceotherpossiblebiochemicalactivitiescouldbe
attributedtotheQQenzymes,thehighprevalencecannotbeunequivocallyrelatedtoAHL
degradation.Inthissense,theMediterraneanmetagenomesanalyzedyieldedahighernumberof
acylasesthanlactonases(Figure4),despitethewidestdiversityofAHLlactonasesidentifiedsofar
[4,39].AhigherprevalenceofacylaseshasalsobeenreportedpreviouslyintheGOSsequences[48],
eventhoughatthattimethenumberofknownlactonaseswasmuchlower.Typically,AHLacylases
areconsideredmoresubstrate‐specificthanlactonasesandhavebeendefinedasexclusiveAHL
degraders.However,therecentidentificationofacylaseswithabroadspecificitywhichcanalso
degradepenicillinG[96,97],hasaddedcomplexitytothemetabolicroleofAHLacylases.This
promiscuityofsubstratescouldpartiallyexplainthishigherfrequencyandquestiontheexclusive
activityoftheseenzymesinthedegradationofAHL‐typeQSsignals.Therefore,anddespitethedata
obtainedfromthemetagenomicanalysisseemtoconfirmthehighestprevalenceofQQactivitiesin
deepersamples,thisresultshouldbeinterpretedwithcautionuntiltheQQactivityofsomeofthe
retrievedsequencescanbeconfirmed.
Inthisstudy,wehavefoundthatthepresenceofthethreetypesofAHLsynthaseswasverylow
intheentirewatercolumn.ThestudyofmetagenomicdatabasesfromtheAtlantic,Pacific,andIndian
Oceans[33]demonstratedthattheQSgenesLuxI,AinS,andespeciallyHdtSarepresentinamuch
widerdiversityofmarinebacteriathanpreviouslysuspectedfromcultivablebacteria,andtheywere
morefrequentlyidentified(0.02–14.8)incomparisontothepresentstudy(0.01–0.17).Asdescribed
hereforthevariabilityintheQQactivity,differencesinnutrientavailabilityorphysicochemical
conditionsmayberesponsibleforthesedifferencesamongsamplingsites,althoughdifferencesin
themethodologyappliedcannotbedisregardedasasignificantsourceofvariation.AHLreceptors
weremuchmorecommonthanAHLsynthasesintheMediterraneansamples,especiallyat2000m
depth(Figure4A),correlatingwithahigherabundanceofQQactivityandgenes.Inagreementwith
thisunbalancebetweenQSsynthasesandreceptors,apreviousstudyshowedthatamongthe68
genomesofProteobacteriacompared,45containedorphanLuxRhomologsbutnoadditionalLuxI
homologs,and66%showedmoreLuxRthanLuxIhomologs[98].Thisunbalancecanbeattributed
toalargeprevalenceoforphanLuxRreceptorsinthebacterialpopulation.Themicroorganismswith
anorphanLuxRdonotregulatedirectlytheAHLsynthesis,buttheycanparticipateinthecell‐to‐
cellbacterialcommunication.Thissupportstheideathattheabilityto“sense”otherbacteriaismore
efficientthanthecapacityto“talk”tothem,sincetheAHL‐productionishighlyenergycostly.
Moreover,othermolecules,suchascinnamoyl‐HSL,p‐coumaroyl‐HSL,α‐pyrones,and
dialkylresorcinols,havebeenreportedtoactivateLuxR‐typereceptors[11].Nevertheless,intheview
ofthelargeunbalancebetweenthenumberofsynthasesandreceptorsidentifiedinthe
Mediterraneanmetagenomes,thehypothesisoftheexistenceofotheryetunknownAHLsynthases
cannotbeexcluded.
Surprisingly,anddespiteVibriosp.representinga3.5%ofthebacterialpopulationinthe
analyzedsamples,noLuxShomologscouldbeidentifiedintheentirewatercolumn,indicatingthat
theAHL‐mediatedQSprocessesaremoreabundantthantheAI‐2signalingpathwayinthemarine
environment.Incoherencewiththisresult,LuxShomologuescouldbeidentifiedinonly3ofthe13
sitesincludedintheGOSdatabase,withanormalizedfrequencyof0.7[33].Thisdataonthe
prevalenceoftheluxSgenecontrastswiththe653sequences(14.8)affiliatedtoHdtSinthesame
study,confirmingthattheAHL‐mediatedQSprocessesaremorefrequentthantheAI‐2signalingin
themarineenvironment.
ThehighpercentageofisolatedstrainswiththeabilitytoactivateAHLbiosensorandtodisrupt
long‐chainAHLQSsignalsintheMediterraneanSea,combinedwiththehighfrequencyofQQand
QSgenesthatcorrelatewithlowerCavailability,indicatesthatQSandQQcouldbeusualstrategies
inthismarinehabitatthatmayconferacompetitiveadvantageinoligotrophicconditions.
Furthermore,theanalysisofthemetagenomicdataindicatesthatQSandQQprocesseshavean
importantroleindeepmarinehabitats.Onsite,ecologicalstudiesonQSandQQactivityandthe
Genes2018,9,100 15of20
identificationofmoreQS‐andQQ‐relatedsequencesinthemarinehabitatswouldprobablyprovide
keyknowledgetofurtherelucidatetheecologicalimportanceofAHL‐mediatedQSandQQprocesses
inthemarineenvironment.
SupplementaryMaterials:Thefollowingareavailableonlineathttp://www.mdpi.com/2073‐4425/9/2/100/s1,
TableS1:Totalisolatedstrainsfromdifferentsamplesandculturemedia,showingthenumberandpercentage
ofstrainswithputativeQSandQQactivityagainstC6‐andC12‐HSLobtainedusingthesolidplateC.violaceum
assay.Mediaused:tryptonesoyagar1%NaCl(TSA‐1),marineagar(MA),dilutedmarineagar(MA1/100),and
filteredautoclavedseawatermedium(FAS)supplementedwith0.5g/Lpolymers:agarose,chitin,andstarch
(FAS‐POL).
Acknowledgments:Thisworkwassupportedbythenon‐profitorganizationsFundaciónRamónAreces
(CIVP16A1814),theEUProjectByefouling(grantagreementno612717),andbythegrant“AxudasdoPrograma
deConsolidacióneEstructuracióndeUnidadesdeInvestigaciónCompetitivas(GPC)”fromtheConselleríade
Cultura,EducacióneOrdenaciónUniversitaria,XuntadeGalicia(ED431B2017/53).A.M.wassupportedbya
predoctoralfellowshipfromtheConselleríadeCultura,EducacióneOrdenaciónUniversitaria,XuntadeGalicia
(ED481A‐2015/311).M.L.P.wassupportedbyaPostdoctoralfellowshipfromtheValencianConselleríade
Educació,Investigació,CulturaiEsport(APOSTD/2016/051).WewouldliketothankPaulWilliamsfrom
UniversityofNottinghamandTomohiroMorohoshifromUtsunomiyaUniversityforkindlyprovidinguswith
theChromobacteriumviolaceumCV026andVIR07biosensorstrains,respectively.
AuthorContributions:A.M.,C.M.,A.P.,J.A.,andA.O.carriedouttheisolation,screening,andselectionofthe
marinestrains.Theyalsocharacterizedandidentifiedthequorumquenchingstrains.M.L.carriedoutthe
metagenomicanalyses.A.M.,M.L.,andA.O.wereinchargeofthewritingofthepaper.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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Supplementary resource (1)

Supplementary Material
Data
February 2018
Andrea Muras · Mario López-Pérez · Celia Mayer · Ana Parga · Ana Otero
... Quorum quenching (QQ) inhibits QS pathways by interfering with the production, release, recognition, and degradation of signaling molecules, thereby affecting various physiological functions dependent on bacterial QS [23,24]. Indeed, bacterial signaling molecules in the microenvironment could be removed by QQ activity [25], which was a crucial process for bacteria to maintain environmental homeostasis [2]. ...
... It was reported that AHL-acylases preferred to degrade AHLs with longer carbon chain and could not degrade C 4 -HSL and C6-HSL in some bacteria [60,61]. Further, AHL acylases in cultivable marine bacteria [3,27,59,62] and in marine metagenomic collections [23,24] seems to be more abundant than AHL lactonases. In our study, more bacteria from sediment samples were found to exhibit QQ activities. ...
Diversity of Bacteria with Quorum Sensing and Quenching Activities from Hydrothermal Vents in the Okinawa Trough
Article
Full-text available
  • Mar 2023
Quorum sensing (QS) is a chemical communication system by which bacteria coordinate gene expression and social behaviors. Quorum quenching (QQ) refers to processes of inhibiting the QS pathway. Deep-sea hydrothermal vents are extreme marine environments, where abundant and diverse microbial communities live. However, the nature of chemical communication in bacteria inhabiting the hydrothermal vent is poorly understood. In this study, the QS and QQ activities with N-acyl homoserine lactones (AHLs) as the autoinducer were detected in bacteria isolated from hydrothermal vents in the Okinawa Trough. A total of 18 and 108 isolates possessed AHL-producing and AHL-degrading abilities, respectively. Bacteria mainly affiliated with Rhodobacterales, Hyphomicrobiales, Enterobacterales and Sphingomonadales showed QS activities; QQ was mainly associated with Bacillales, Rhodospirillales and Sphingomonadales. The results showed that the bacterial QS and QQ processes are prevalent in hydrothermal environments in the Okinawa Trough. Furthermore, QS significantly affected the activities of extracellular enzymes represented by β-glucosidase, aminopeptidase and phosphatase in the four isolates with higher QS activities. Our results increase the current knowledge of the diversity of QS and QQ bacteria in extreme marine environments and shed light on the interspecific relationships to better investigate their dynamics and ecological roles in biogeochemical cycling.
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... Enzymatic degradation is often displayed as a lack of pigmented halo or as a halo of reduced diameter respect to the negative control. On the other hand, QS inhibitors diffuse with the AHL added to the sample; the different titers of both components, with the AHL normally present in a much higher titer, result in an inner non-pigmented halo surrounded by an outer violet halo (Muras et al., 2018a). Furthermore, this assay allows differentiating between QS inhibition (observation of growth-turbid halos) and cell growth inhibition (observation of transparent halos) (Muras et al., 2018a). ...
... On the other hand, QS inhibitors diffuse with the AHL added to the sample; the different titers of both components, with the AHL normally present in a much higher titer, result in an inner non-pigmented halo surrounded by an outer violet halo (Muras et al., 2018a). Furthermore, this assay allows differentiating between QS inhibition (observation of growth-turbid halos) and cell growth inhibition (observation of transparent halos) (Muras et al., 2018a). ...
The quorum quenching enzyme Aii20J modifies in vitro periodontal biofilm formation
Article
Full-text available
  • Feb 2023
Introduction Recent studies have revealed the presence of N-acyl-homoserine lactones (AHLs) quorum sensing (QS) signals in the oral environment. Yet, their role in oral biofilm development remains scarcely investigated. The use of quorum quenching (QQ) strategies targeting AHLs has been described as efficient for the control of pathogenic biofilms. Here, we evaluate the use of a highly active AHL-targeting QQ enzyme, Aii20J, to modulate oral biofilm formation in vitro. Methods The effect of the QQ enzyme was studied in in vitro multispecies biofilms generated from oral samples taken from healthy donors and patients with periodontal disease. Subgingival samples were used as inocula, aiming to select members of the microbiota of the periodontal pocket niche in the in vitro biofilms. Biofilm formation abilities and microbial composition were studied upon treating the biofilms with the QQ enzyme Aii20J. Results and Discussion The addition of the enzyme resulted in significant biofilm mass reductions in 30 – 60% of the subgingival-derived biofilms, although standard AHLs could not be found in the supernatants of the cultured biofilms. Changes in biofilm mass were not accompanied by significant alterations of bacterial relative abundance at the genus level. The investigation of 125 oral supragingival metagenomes and a synthetic subgingival metagenome revealed a surprisingly high abundance and broad distribution of homologous of the AHL synthase HdtS and several protein families of AHL receptors, as well as an enormous presence of QQ enzymes, pointing to the existence of an intricate signaling network in oral biofilms that has been so far unreported, and should be further investigated. Together, our findings support the use of Aii20J to modulate polymicrobial biofilm formation without changing the microbiome structure of the biofilm. Results in this study suggest that AHLs or AHL-like molecules affect oral biofilm formation, encouraging the application of QQ strategies for oral health improvement, and reinforcing the importance of personalized approaches to oral biofilm control.
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... Consequently, these findings suggest that in this particular strain, bioluminescence regulation may not be density-dependent and could be independent of conventional QS control mechanisms. Expanding the scope, another study by Muras et al. [25] assessed the prevalence of QS in deep-sea environments. The study found that deep-sea samples collected at a depth of 2000 meters had a comparatively low abundance of bacterial members involved in QS than surface samples from a depth of 15 meters. ...
Quorum Sensing: Unravelling the Intricacies of Microbial Communication for Biofilm Formation, Biogeochemical Cycling, and Biotechnological Applications
Article
Full-text available
  • Aug 2023
The marine environment possesses diverse and complex characteristics, representing a significant challenge for microbial survival. Therefore, bacteria must develop adaptive mechanisms to thrive in such environments. Quorum sensing (QS), a well-established phenomenon in microorganisms, involves the communication between cells through chemical signals, which is dependent on cell density. Extensive research has been conducted on this microbial ability, encompassing the early stages of understanding QS to the latest advancements in the identification and characterization of its mechanisms. This minireview comprehensively examines the role of QS in various aspects, including biofilm formation, virulence in pathogenic bacteria, such as Vibrio spp. And Pseudomonas spp., as well as its influence on biogeochemical cycling in deep-sea environments. Furthermore, future progress in the field will be achieved by combining state-of-the-art methods for observing QS in the deep sea with a deeper understanding of the underlying processes, which will facilitate the engineering of microorganisms for improved degradation of persistent environmental pollutants and other biotechnological applications.
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... For biofilm supernatants treated with Aii20J at a titer of 20 µg/mL, 100 µL was transferred to a fresh microtube at the end of the incubation time. Both the purified Aii20J and biofilm supernatants containing Aii20J were incubated with 10 µM of C 6 -HSL for 3 h at 22 • C. PBS (pH 6.5) with 10 µM of C 6 -HSL was used as a negative control [47]. The bioassays were carried out as described elsewhere [27]. ...
New Preventive Strategy against Oral Biofilm Formation in Caries-Active Children: An In Vitro Study
Article
Full-text available
  • Jul 2023
Quorum quenching (QQ) is the inhibition of bacterial communication, i.e., quorum sensing (QS). QS is a key mechanism in regulating biofilm formation and phenotype in complex bacterial communities, such as those found within cariogenic biofilms. Whereas QQ approaches were shown to effectively reduce biomass, knowledge of their impact on the taxonomic composition of oral polymicrobial biofilms remains scarce. Here, we investigate the effect of the QQ lactonase Aii20J on biomass production and taxonomical composition of biofilms. We collected supragingival plaque samples from 10 caries-free and 10 caries-active children and cultured them to generate in vitro biofilms. We describe significant biomass reductions upon Aii20J exposure, as assessed by crystal violet assays. Taxonomical profiling using 16S rRNA gene amplicon sequencing revealed no significant changes in bacterial composition at the genus level. Interestingly, at the species level Aii20J-treatment increased the abundance of Streptococcus cristatus and Streptococcus salivarius. Both S. cristatus and S. salivarius express pH-buffering enzymes (arginine deiminase and urease, respectively) that catalyze ammonia production, thereby potentially raising local pH and counteracting the biofilm’s cariogenic potential. Within the limitations of the study, our findings provide evidence of the biofilm-modulating ability of QQ and offer novel insights into alternative strategies to restore homeostasis within dysbiotic ecosystems.
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... These findings led them to conclude that the deep-sea strain of Photobacterium phosphoreum ANT-2200 is not density-dependent and, therefore, not susceptible to quorum sensing control. Another study by Muras et al. [24] investigated the prevalence of quorum sensing in deep-sea environments. The researchers found that the abundance of bacterial members involved in quorum sensing was relatively low in deep-sea samples collected at a depth of 2000 meters compared to surface samples taken at 15 meters. ...
Quorum Sensing: Unravelling the Intricacies of Microbial Communication for Biofilm Formation, Biogeochemical Cycling, and Biotechnological Applications
Preprint
Full-text available
  • Jul 2023
Quorum sensing (QS), a well-established phenomenon in microorganisms, involves the communication between cells through chemical signals, which is dependent on cell density. Extensive research has been conducted on this microbial ability, encompassing the early stages of understanding QS to the latest advancements in the identification and characterization of its mechanisms. This minireview comprehensively examines the role of QS in various aspects, including biofilm formation, virulence in pathogenic bacteria such as Helicobacter pylori and Pseudomonas sp., as well as its influence on biogeochemical cycling in deep-sea environments. Furthermore, future progress in the field will be achieved by combining state-of-the-art methods for observing QS in the deep sea with a deeper understanding of the underlying processes, which will facilitate the engineering of microorganisms for improved degradation of persistent environmental pollutants and other biotechnological applications.
View
... Both the purified Aii20J and biofilm supernatants containing Aii20J were incubated with 10 μM of C6-HSL for 3 hours at 22 °C. PBS (pH 6.5) with 10 μM of C6-HSL was used as negative control [44]. The bioassays were carried out as described elsewhere [27]. ...
New Preventive Strategy Against Oral Biofilm Formation In Caries-Active Children: An In Vitro Study
Preprint
Full-text available
  • Jun 2023
Caries are lesions caused by acidic compounds derived from the metabolism of bacteria such as streptococci. It is not merely the presence of acidogenic bacteria that causes caries but an imbalance in the oral biofilm microhabitat and its behavior. One factor underlying biofilm formation is quorum sensing (QS), that in gram-positives relies on peptidic molecules, whereas in gram-negatives is driven by small diffusible signals. Strategies based on QS inhibition have been proposed as alter-natives to antimicrobial therapies. Here, we investigate the antibiofilm potential of the lactonase enzyme Aii20J, previously reported as effective against biofilms of periodontal disease origin. We generated in vitro polymicrobial biofilms dominated by gram-positive taxa using supragingival samples from caries-free and caries-active children. The effect of Aii20J on the biofilms was evalu-ated regarding its biomass-reducing ability, as assessed with crystal violet assay, and its effects on the bacterial composition of the polymicrobial biofilms, as assessed by 16S community profiling. We describe significant biomass reductions upon Aii20J exposure without significant changes in bac-terial composition at the genus level. Our results support the use of Aii20J to prevent oral biofilm formation while highlighting the influence of the polymicrobial environment on bacterial commu-nities and their response to antibiofilm treatments.
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... Researchers have isolated microorganisms capable of QS in diverse biofilms colonizing terrestrial and aquatic environments in either temperate or extreme ecosystems. Additionally, researchers have identified QS genes in metagenomic-based environmental studies (Riaz et al. 2008;Doberva et al. 2015;Muras et al. 2018). The importance of QS in the functionality of microbial loops in oceans and, more generally, in the functionality of aquatic and terrestrial ecosystems is a recent but growing research topic (Hmelo 2017;Urvoy et al. 2022). ...
Quorum sensing in biofilms: a key mechanism to target in ecotoxicological studies
Article
Our environment is heavily contaminated by anthropogenic compounds, and this issue constitutes a significant threat to all life forms, including biofilm-forming microorganisms. Cell–cell interactions shape microbial community structures and functions, and pollutants that affect intercellular communications impact biofilm functions and ecological roles. There is a growing interest in environmental science fields for evaluating how anthropogenic pollutants impact cell–cell interactions. In this review, we synthesize existing literature that evaluates the impacts of quorum sensing (QS), which is a widespread density-dependent communication system occurring within many bacterial groups forming biofilms. First, we examine the perturbating effects of environmental contaminants on QS circuits; and our findings reveal that QS is an essential yet underexplored mechanism affected by pollutants. Second, our work highlights that QS is an unsuspected and key resistance mechanism that assists bacteria in dealing with environmental contamination (caused by metals or organic pollutants) and that favors bacterial growth in unfavourable environments. We emphasize the value of considering QS a critical mechanism for monitoring microbial responses in ecotoxicology. Ultimately, we determine that QS circuits constitute promising targets for innovative biotechnological approaches with major perspectives for applications in the field of environmental science.
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... AHL-mediated QS were significantly higher in low depth of 90 m (37.66%) than 2000 m (4.01%). On the contrary, genes associated with QQ were 1.63-folds more in the 2000 m (Muras et al., 2018). Interestingly, exploration through culturable and metagenomic approaches indicates that QQ genes are as common as nutrient acquisition genes in the marine microbiome, which contrasts with the low stability of AHLs in marine saline water, suggesting that QQ enzymes could be promiscuous (Romero et al., 2012). ...
Metagenomic insights into taxonomic, functional diversity and inhibitors of microbial biofilms
Article
  • Sep 2022
  • MICROBIOL RES
Microbial cells attached to inert or living surfaces adopt biofilm with self-produced exopolysaccharide matrix containing polysaccharides, proteins, and extracellular DNA, for protection from adverse external stimuli. Biofilms in hospitals and industries serve as a breeding ground for drug-resistant pathogens and ARG enrichment that are linked to pathogenicity and also impede industrial production process. Biofilm formation, including virulence and pathogenicity, is regulated through quorum sensing (QS), a means of bacterial cell to cell communication for cooperative physiological processes. Hence, QS inhibition through quorum quenching (QQ) is a feasible approach to inhibit biofilm formation. In contrast, biofilms have beneficial roles in promoting plant growth, biocontrol, and wastewater treatment. Furthermore, polymicrobial biofilms can harbour novel compounds and species of industrial and pharmaceutical interest. Hence, surveillance of biofilm microbiome structure and functional attributes is crucial to determine the extent of the risk it poses and to harness its bioactive potential. One of the most preferred approaches to delineate the microbiome is culture-independent metagenomics. In this context, this review article explores the biofilm microbiome in built and natural settings such as agriculture, household appliances, wastewater treatment plants, hospitals, microplastics, and dental biofilm. We have also discussed the recent reports on discoveries of novel QS and biofilm inhibitors through conventional, metagenomics, and machine learning approaches. Finally, we present biofilm-derived novel metagenome-assembled genomes (MAGs), genomes, and taxa of medical and industrial interest.
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Activités enzymatiques extracellulaires et composition des communautés bactériennes dans l’estuaire de l’Aulne et la rade de Brest : facteurs de régulation et rôle du quorum sensing
Thesis
  • May 2022
Les bactéries hétérotrophes jouent un rôle primordial dans le fonctionnement des écosystèmes marins. Cette thèse s’est intéressée aux facteurs influençant la composition des communautés bactériennes estuariennes et marines ainsi que leurs fonctions dans la dégradation de la matière organique. Pour cela, une étude a été réalisée au sein de l’estuaire de l’Aulne et des eaux côtières adjacentes de la rade de Brest. Des expériences en laboratoire ont permis d’appréhender le rôle spécifique des communications bactériennes basées sur le quorum sensing (QS). Les résultats obtenus ont montré que l’estuaire de l’Aulne était un véritable bioréacteur naturel. Il abrite des communautés bactériennes dont la composition et le niveau d’activité enzymatique suivent de fortes variations spatiotemporelles, liées aux conditions physicochimiques mais également à des facteurs biotiques comme leur degré de concurrence.Ils ont également démontré la forte implication du QS dans la régulation de la synthèse d’enzymes hydrolytiques au sein de souches isolées et de communautés bactériennes naturelles. Ce mécanisme de régulation peut aussi impacter la composition des communautés bactériennes en régulant les processus liés à la colonisation et aux interactions microbiennes, qu’elles soient trophiques ou non. Dans son ensemble, ce travail de thèse met en avant l’importance de considérer les facteurs biotiques dans l’étude des communautés bactériennes naturelles. Il souligne aussi la nécessité de mieux caractériser les interactions microbiennes, de leur mise en place d’un point de vue moléculaire à leurs impacts biogéochimiques globaux au sein des écosystèmes marins.
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Fine stratification of microbial communities through a metagenomic profile of the photic zone
Preprint
Full-text available
  • May 2017
Most marine metagenomic studies of the marine photic zone analyze only samples taken at one or two depths. However, when the water column is stratified, physicochemical parameters change dramatically over relatively short depth intervals. We sampled the photic water column every 15m depth at a single point of an off-shore Mediterranean site during a period of strong stratification (early autumn) to evaluate the effects of small depth increases on the microbiome. Using genomic assembly and metagenomic read recruitment, we found major shifts in the community structure over small variations of depth, with most microbes showing a distribution limited to layers approximately 30 meters thick (stenobathic). Only some representatives of the SAR11 clade and the Sphingomonadaceae appeared to be eurybathic, spanning a greater range of depths. These results were confirmed by studying a single gene (rhodopsin) for which we also found narrow depth distributions. Our results highlight the importance of considering vertical distribution as a major element when analyzing the presence of marine clades and species or comparing the microbiome present at different locations.
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Genome diversity of marine phages recovered from Mediterranean metagenomes: Size matters
Article
Full-text available
Marine viruses play a critical role not only in the global geochemical cycles but also in the biology and evolution of their hosts. Despite their importance, viral diversity remains underexplored mostly due to sampling and cultivation challenges. Direct sequencing approaches such as viromics has provided new insights into the marine viral world. As a complementary approach, we analysed 24 microbial metagenomes (>0.2 μm size range) obtained from six sites in the Mediterranean Sea that vary by depth, season and filter used to retrieve the fraction. Filter-size comparison showed a significant number of viral sequences that were retained on the larger-pore filters and were different from those found in the viral fraction from the same sample, indicating that some important viral information is missing using only assembly from viromes. Besides, we were able to describe 1,323 viral genomic fragments that were more than 10Kb in length, of which 36 represented complete viral genomes including some of them retrieved from a cross-assembly from different metagenomes. Host prediction based on sequence methods revealed new phage groups belonging to marine prokaryotes like SAR11, Cyanobacteria or SAR116. We also identified the first complete virophage from deep seawater and a new endemic clade of the recently discovered Marine group II Euryarchaeota virus. Furthermore, analysis of viral distribution using metagenomes and viromes indicated that most of the new phages were found exclusively in the Mediterranean Sea and some of them, mostly the ones recovered from deep metagenomes, do not recruit in any database probably indicating higher variability and endemicity in Mediterranean bathypelagic waters. Together these data provide the first detailed picture of genomic diversity, spatial and depth variations of viral communities within the Mediterranean Sea using metagenome assembly.
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Quorum Sensing and Quorum Quenching in the Phycosphere of Phytoplankton: a Case of Chemical Interactions in Ecology
Article
Full-text available
  • Dec 2016
  • J CHEM ECOL
The interactions between bacteria and phytoplankton regulate many important biogeochemical reactions in the marine environment, including those in the global carbon, nitrogen, and sulfur cycles. At the microscopic level, it is now well established that important consortia of bacteria colonize the phycosphere, the immediate environment of phytoplankton cells. In this microscale environment, abundant bacterial cells are organized in a structured biofilm, and exchange information through the diffusion of small molecules called semiochemicals. Among these processes, quorum sensing plays a particular role as, when a sufficient abundance of cells is reached, it allows bacteria to coordinate their gene expression and physiology at the population level. In contrast, quorum quenching mechanisms are employed by many different types of microorganisms that limit the coordination of antagonistic bacteria. This review synthesizes quorum sensing and quorum quenching mechanisms evidenced to date in the phycosphere, emphasizing the implications that these signaling systems have for the regulation of bacterial communities and their activities. The diversity of chemical compounds involved in these processes is examined. We further review the bacterial functions regulated in the phycosphere by quorum sensing, which include biofilm formation, nutrient acquisition, and emission of algaecides. We also discuss quorum quenching compounds as antagonists of quorum sensing, their function in the phycosphere, and their potential biotechnological applications. Overall, the current state of the art demonstrates that quorum sensing and quorum quenching regulate a balance between a symbiotic and a parasitic way of life between bacteria and their phytoplankton host. Keywords Quorum sensingQuorum quenchingPhytoplanktonPhycosphere
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Links between Transcription, Environmental Adaptation and Gene Variability in Escherichia coli : Correlations between Gene Expression and Gene Variability Reflect Growth Efficiencies
Article
Full-text available
  • Jun 2016
  • MOL BIOL EVOL
Gene expression is known to be the principle factor explaining how fast genes evolve. Highly transcribed genes evolve slowly because any negative impact caused by a particular mutation is magnified by protein abundance. However, gene expression is a phenotype that depends both on the environment and on the strains or species. We studied this phenotypic plasticity by analyzing the transcriptome profiles of four Escherichia coli strains grown in three different culture media, and explored how expression variability was linked to gene allelic diversity. Genes whose expression changed according to the media and not to the strains were less polymorphic than other genes. Genes for which transcription depended predominantly on the strain were more polymorphic than other genes and were involved in sensing and responding to environmental changes, with an overrepresentation of two-component system genes. Surprisingly, we found that the correlation between transcription and gene diversity was highly variable among growth conditions and could be used to quantify growth efficiency of a strain in a medium. Genetic variability was found to increase with gene expression in poor growth conditions. As such conditions are also characterized by down-regulation of all DNA repair systems, including transcription-coupled repair, we suggest that gene expression under stressful conditions may be mutagenic and thus leads to a variability in mutation rate among genes in the genome which contributes to the pattern of protein evolution.
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Selection of the N-Acylhomoserine Lactone-Degrading Bacterium Alteromonas stellipolaris PQQ-42 and of Its Potential for Biocontrol in Aquaculture
Article
Full-text available
  • May 2016
The production of virulence factors by many pathogenic microorganisms depends on the intercellular communication system called quorum sensing, which involves the production and release of signal molecules known as autoinducers. Based on this, new-therapeutic strategies have emerged for the treatment of a variety of infections, such as the enzymatic degradation of signaling molecules, known as quorum quenching (QQ). In this study, we present the screening of QQ activity amongst 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of the strain PQQ-42, which degrades a wide range of N-acylhomoserine lactones (AHLs). The selected strain, identified as Alteromonas stellipolaris, degraded the accumulation of AHLs and reduced the production of protease and chitinase and swimming motility of a Vibrio species in co-cultivation experiments in vitro. In the bio-control experiment, strain PQQ-42 significantly reduced the pathogenicity of Vibrio mediterranei VibC-Oc-097 upon the coral Oculina patagonica showing a lower degree of tissue damage (29.25 ± 14.63%) in its presence, compared to when the coral was infected with V. mediterranei VibC-Oc-097 alone (77.53 ± 13.22%). Our results suggest that this AHL-degrading bacterium may have biotechnological applications in aquaculture.
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Phylogenetic identification and in situ detection of individual microbial cells without cultivation
Article
  • Mar 1995
  • Microbiol Rev
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
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tRNAscan-SE: A program for improved detection of transfer RNA Genes in genomic sequence
Article
  • Jan 1997
We describe a program, tRNAscan-SE, which identifies 99-100% of transfer RNA genes in DNA sequence while giving less than one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free grammars. tRNAscan-SE searches at approximately 30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudogenes.
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Quorum Sensing in Marine Microbial Environments
Article
  • Oct 2016
  • ANNU REV MAR SCI
Quorum sensing (QS) is a form of chemical communication used by certain bacteria that regulates a wide range of biogeochemically important bacterial behaviors. Although QS was first observed in a marine bacterium nearly four decades ago, only in the past decade has there been a rise in interest in the role that QS plays in the ocean. It has become clear that QS, regulated by signals such as acylated homoserine lactones (AHLs) or furanosyl-borate diesters [autoinducer-2 (AI-2) molecules], is involved in important processes within the marine carbon cycle, in the health of coral reef ecosystems, and in trophic interactions between a range of eukaryotes and their bacterial associates. The most well-studied QS systems in the ocean occur in surfaceattached (biofilm) communities and rely on AHL signaling. AHL-QS is highly sensitive to the chemical and biological makeup of the environment and may respond to anthropogenic change, including ocean acidification and rising sea surface temperatures. Expected final online publication date for the Annual Review of Marine Science Volume 9 is January 03, 2017. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
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