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AQUATICMICROBIALECOLOGYAquatMicrobEcolVol.
76:133–147,2015doi:10.
3354/ame01772PublishedonlineOctober22INTRODUCTIONDimethylsulfoniopropionate(DMSP)isatertiarysulfoniumcompoundsynthesizedbymanyspeciesofmarinephytoplanktonandsomemacroalgae.
DMSPisanimportantcomponentofthesulfurandcarboncycleswithinthemicrobialfoodweb,andalsohasseveralecophysiologicalfunctionswithinthemicro-bialcommunity.
Producedatintracellularconcentra-tionstypicallyrangingfrom1to400mM(Kelleretal.
1989,Stefels2000),DMSPsynthesiscontributesupto10%ofallcarbonfixedbyprimaryproduction(Matrai&Keller1994,Archeretal.
2001,Howardetal.
2006).
Whiletheevolutionarydriversleadingtotheallocationofsuchalargefractionofthealgae'sresourcesintoasinglecompoundarestillunclear,itiswellknownthatseveralmicro-andmacroalgaeuseitasacompatiblesolutetocopewithosmoticandthermalstresses(Dicksonetal.
1980,Reed1983,Dickson&Kirst1986,1987,Kirstetal.
1991,Karstenetal.
1992,Nishiguchi&Somero1992).
AdditionalfunctionsforDMSPincludeprotectionagainstoxida-tivestress,withDMSPanditsdegradationproductseffectivelyscavengingreactiveoxygenspeciesInter-Research2015·www.
int-res.
com*Correspondingauthor:jmotard-cote@disl.
orgOsmoprotectiveroleofdimethylsulfoniopropionate(DMSP)forestuarinebacterioplanktonJessieMotard-Cté1,2,*,RonaldP.
Kiene1,21DepartmentofMarineSciences,UniversityofSouthAlabama,Mobile,Alabama36688,USA2DauphinIslandSeaLab,101BienvilleBlvd,DauphinIsland,Alabama36528,USAABSTRACT:Dimethylsulfoniopropionate(DMSP)issynthesizedandusedbymarinephyto-planktonasanosmolyte.
PreviousstudieshaveshownthatsomeofthedissolvedDMSP(DMSPd)inseawateristakenupbybacterioplanktonandnotdegraded.
Wetestedthehypoth-esisthatretentionofDMSPprovidessomebenefitstomarinebacteria.
Inexperimentswithcoastalseawaterfiltratescontainingmainlybacteria,acuteosmoticstressesof+5and+10pptNaClsignificantlyinhibitedbacterialproduction(BP)over6h,whiletheavailabilityof20nMDMSPdrelievedmostoftheBPinhibition.
Partialreliefofsalt-inducedinhibitionofBPwasobservedwithDMSPdconcentrationsaslowas2.
5nM,andDMSPwasmoreeffectiveatre-lievingosmoticstressthanothercompoundstested.
OsmoticstressesresultedinafasterandgreateroveralluptakeofDMSPdandaccumulationofuntransformedDMSPinbacterialcells(DMSPcell).
RetainedDMSPreachedosmotically-significantintracellularconcentrationsof54mMinsalt-stressedbacterialpopulations.
RetentionofDMSPwasaccompaniedbyalowerproductionofmethanethiol(MeSH),suggestingadown-regulationofthedemethylation/demethiolationpathwayunderosmoticstress.
TheseresultsshowthatestuarinebacterioplanktoncanuseDMSPasanosmoprotectant,retainingupto54%oftheavailabledissolvedDMSPun-transformedintheircells.
ThisbenefitprovidedbyDMSPmayhelpexplainwhysomeDMSPisretainedinbacteriaintheocean,evenunderunchangingsalinity.
ThisretentionslowsdownthecyclingofDMSP,withpotentialimplicationsforthetrophictransferofDMSPanditscontributionstosulfurandcarbonfluxesintheocean.
KEYWORDS:Osmolyte·Compatiblesolute·Salinitystress·Saltinhibition·Osmolarity·Sulfurcycle·DMSPretention·DMSPfunctionResaleorrepublicationnotpermittedwithoutwrittenconsentofthepublisherAquatMicrobEcol76:133–147,2015(Sundaetal.
2002),andoverflowmetabolism,allow-ingphotosyntheticcellstodissipateexcessreducedcompoundsandenergyundernutrientlimitationorexcessivecarbonfixationrates,preventingpotentialnegativefeedbacksonmetabolicpathways(Stefels2000).
Afewecologicalfunctionshavealsobeenpro-posedforDMSP,suchasbeingagrazingdeterrent(Wolfeetal.
1997,VanAlstyneetal.
2001,Strometal.
2003)andchemoattractantformarinebacteria(Zimmer-Faustetal.
1996,Milleretal.
2004,Sey-mouretal.
2010).
Mostofthephytoplankton-DMSPloss(63to91%)ismediatedbymicrozooplanktongrazing(Archeretal.
2002,Simóetal.
2002).
IngestedDMSPcaneitherbereleasedasdissolvedDMSPorasdimethylsulfide(DMS)uponconversionbybacterialoralgalDMSPlyases,oritcanberetainedandeitherpassedupthefoodchainorassimilatedtosatisfysulfurrequire-ments.
About30%ofthephytoplankton-DMSPin-gestedbyherbivorousprotistsisretainedintheircells(Simóetal.
2002,Tang&Simó2003,Salóetal.
2009).
Thisretentionseemstobetemporary,andDMSP-sulfuriseventuallyassimilatedintothegrazer'sbiomass(Belvisoetal.
1990,Salóetal.
2009).
However,someDMSPcanbetransferredtohighertrophiclevels,presumablywhenDMSP-bearingpro-tistsareeatenbeforecompletingDMSPtransfor-mation.
DMSPhasbeenmeasuredinseveralomniv-orousandcarnivorouscopepodspeciesaftergutclearance,suggestingthatthisDMSPwaslocatedinbodytissues(Tangetal.
1999),makingitmorelikelytobetransferredtohighertrophiclevels.
DMSPhasalsobeenfoundintissuesofseveralotheranimals(bivalves,gastropods,crustaceansandfish)thatmostlikelyacquireditfromingestedfood(Tokunagaetal.
1977,Iida&Tokunaga1986,Daceyetal.
1994,Hilletal.
2000).
ThefactthatDMSPhasevenbeenfoundincarnivorousfishessuchascod(Levasseuretal.
1994),salmon(Motohiro1962)andmackerel(Ackmanetal.
1972)furtherindicatesthatDMSPcanbetransferredthroughmorethanonetrophiclevel.
Whenreleasedtotheextracellularenvironment,DMSPisrapidlytakenupanddegradedbyhetero-trophicprokaryotesforenergyandbiomass,usingoneof2alternativepathways.
Thedemethylation/demethiolationpathwayultimatelyproducesmeth-anethiol(MeSH;Kiene&Taylor1988),someofwhichisrapidlyincorporatedintomacromolecules(Gon-zálezetal.
1999,Kieneetal.
1999).
Thispathwayiswidespreadamongstbacterioplanktoninthesurfaceocean(Reischetal.
2011),highlightingtheimpor-tanceofDMSPdegradationforassimilationofre-ducedcarbonandsulfurbymarineheterotrophs.
Moreover,biogeochemicaldatashowthatDMSPcansatisfyasmuchas15and100%ofthebacterialrequirementsforcarbonandsulfur,respectively(Kiene&Linn2000a,Zubkovetal.
2001,Simóetal.
2002).
ThesecondpathwayismediatedbyDMSPlyases,whichcleaveDMSPtoDMSandacrylate(Yoch2002,Toddetal.
2007).
DMSemissionsfromtheoceancontributebetween0.
55and1.
1Tmolsul-furyr1totheglobalatmosphere(Lanaetal.
2011)andarehypothesizedtohaveacoolingeffectontheclimatebecauseDMSisoxidizedtoacidicproductsthateitherformoraddontoaerosolparticles(Charl-sonetal.
1987,Quinn&Bates2011).
DMSventilationacrosstheseaairboundaryisalsoamajormecha-nismfortransferringsulfurfromtheoceanstothecontinentsthroughtheatmosphere(Lovelocketal.
1972).
Addingtothesewell-documentedfunctions,thereisgrowingevidencethatDMSPcanserveasanosmolyteforbacteria.
Tomaintainorrestoretheirosmoticbalanceunderhighorchangingsalinity,marineprokaryotesneedtoaccumulateintracellu-larcompatiblesolutes,whichtheycaneithersyn-thesizedenovoortakeupfromtheirenvironment(Galinski1995).
Glycinebetaine(GBT),astructuralanalogueofDMSP,iswellrecognizedasoneofthemostefficientandwidespreadosmolytesusedbyanimals,plantsandbacteria(Yanceyetal.
1982andreferencestherein,Larsenetal.
1987,Anthonietal.
1991andreferencestherein),includingnatu-ralbacterialpopulations(Kiene&HoffmannWilliams1998).
MostmarinebacteriacantakeupGBTandDMSPfromtheenvironmentatnanomolarconcentrations,usingthesamehigh-affinitytrans-portsystem(Kieneetal.
1998).
Previousstudiesfocusingonprokaryotesshowedthatundercon-trolledconditions,availabilityofDMSPpromotesgrowthinhypersalinemediumforsinglestraincul-turesofEscherichiacoli(Chambersetal.
1987,Cosqueretal.
1999)andsomemarinebacteriaiso-lates(Diazetal.
1992,Wolfe1996).
However,whetherwholeassemblagesofmarinebacterio-planktoncantakeupandretainDMSPtocopewithosmoticstressinnaturalsystemshasnotbeenshown.
TogainosmoprotectionfromDMSP,themarinebacterioplanktonwouldhavetoretaintheDMSPuntransformedintheircellsforatleastafewhours,preventingoratleastdelayingitsdegrada-tionthroughthenormalpathways.
Infact,during35S-DMSPduptakeexperimentswithmarinebacte-rioplanktonfromtheGulfofMexico,2.
2%oftheaddedDMSPwasfounduntransformedincells12hafterthetraceraddition(Kiene&Linn2000a).
In134Motard-Cté&Kiene:DMSPasosmoprotectantforbacteria135similarexperimentswithseawatercollectednearHalifax,NovaScotia,Kiene&Linn(2000b)found15%ofaddedDMSPuntransformedinparticlesafter23h.
IntheMediterraneanSea,Belvisoetal.
(1993)measured9.
5%ofparticulateDMSP+DMSinthe0.
2to0.
7msizefraction(correspondingto3.
6nMDMSP+DMS).
However,atthepresenttimeitisnotclearwhetherretentionofDMSPinbacter-ialcellsprovidesanybenefittothosecells.
PreviousstudiesdemonstratingtheuptakeanduseofDMSPasanosmolytebyheterotrophicbacteriawereconductedundersomewhatartificialcondi-tions,usingmonospeciesculturesandhighlevelsofDMSP(≥100nM).
Inthepresentstudy,naturalbacte-rialassemblagesfromcoastalwatersweretestedfortheirabilitytotakeupandretainDMSPuntrans-formedintheircellsunderinducedsalinitystress.
BacterialproductionwasmeasuredtoexaminewhethertheretentionofDMSPenabledthecellstoalleviatethesalt-inducedinhibitionandrestorenor-malcellfunctioning.
Toourknowledge,ourstudyisthefirsttotestwhethernaturalbacterialcommuni-tiesareabletouseDMSPforosmoprotectionpur-posesatthelownanomolarlevelstypicallyfoundinnaturalmarinesystems.
MATERIALSANDMETHODSSamplecollection,preparationandsalinitystressexperimentsSurfacewatersampleswerecollectedfromtheDauphinIslandSeaLab(DISL)pierinMobileBay(GulfofMexico)fromFebruarytoAugust2014.
SalinityattheDISLpieroverthesamplingperiodwashighlyvariable,andsampledwaterrangedfrom10to25partsperthousand(ppt)forthedif-ferentexperiments.
Sampleswerecollectedusinganacid-cleanedbucketandimmediatelybroughtbacktothelaboratory.
Formostexperiments,sea-watersamplesweregravity-filteredthrough142mmdiameterWhatmanGF/Ffiltersusingapoly-carbonatein-linefilterholder(Geotech).
TheGF/Ffilterremovednearlyallofthephytoplanktoncom-munity,butallowedpassageofsomebacteria(typi-cally50to80%;Leeetal.
1995).
Thefiltratewaskeptinthedarkatroomtemperaturefor1to4dtoallowfordepletionofthebioavailabledissolvedDMSP(DMSPd)to0.
2m)withthetotalup-takereachingamaximumofabout48%oftheaddedradiolabelafter0.
8dofincubation(Fig.
1).
Asignifi-cantfraction(19%)ofthemaximumtotaluptake,equivalenttoabout9%oftheadded35S-DMSP,wasretaineduntransformedinthecellsforatleast2d.
Evenafter6dofincubation,therewasstill2.
4%oftheadded35S-DMSPremaininguntransformedinthecells.
Theseresultsindicatethataportionofthenat-uraldissolvedDMSPinseawateristakenupandretainedinmicrobialcellsforlongperiods,evenwhensalinityisstable.
ToaddressthequestionofwhetherDMSPretentionmighthavesomebenefitsforheterotrophicbacteria,wecarriedoutexperi-mentswithseawaterfiltratesinwhichacutesalinitystresswasimposed.
EffectofsalinitystressandDMSPavailabilityonbacterialproductionIna2dpre-incubatedseawaterfiltratecontainingbacteriaandverylowdissolvedDMSPconcentra-tions(0.
2minunfilteredestuarinewaterfromMobileBay(15pptsalinity).
Totaluptakerepre-sentsthe35S-activityinparticlesretainedon0.
2mNylonfilters.
Parallelfiltersateachtimepointweretreatedwith1MNaOH,andthenon-hydrolyzablefractionrepresentstheremaining35Sactivityonthefiltersafteralkalinetreat-ment(non-DMSP35S).
Thedifferencebetweentotaluptakeandnon-hydrolyzable35SrepresentsuntransformedDMSPinparticles>0.
2mFig.
2.
Bacterialproductionrateatambientsalinity(10ppt),+5and+10pptsalinityinaGF/Ffiltrateincubationwithoutdi-methylsulfoniopropionate(DMSP)andwith20nMDMSPadded(A)2hand(B)6haftersaltandDMSPadditions.
ErrorbarsrepresentSDMotard-Cté&Kiene:DMSPasosmoprotectantforbacteria(Fig.
4B).
TheamountofDMSPcell(expressedinnMdissolvedequivalents)after6hwas1.
13nMfortheambientsalinitycontrol,andincreasedto3.
19and4.
56nMforthe+5and+10ppttreatments(ANOVA,p0.
2m;(B)fractionofconsumedDMSPconvertedtodimethylsulfide(DMS);(C)fractionofconsumedDMSPconvertedtomethanethiol(MeSH).
ErrorbarsrepresentSD.
Theseawaterfiltratewaspre-incubatedtoallowendoge-nousDMSPtobeconsumedtolowlevels(<1nM).
ThetimecoursestartedwiththeadditionofNaCl,20nMDMSP,andaspikeoftracer-level35S-DMSPAquatMicrobEcol76:133–147,2015142significantfractionoftheDMSPtakenupwasretaineduntransformedinthecells.
ThisobservationisconsistentwithresultsfromKiene&Linn(1999),whoshowedthatafterincubatingGF/F-filteredseawaterwith35S-DMSPfor25h,11%ofthe35Srecoveredintheparticulatefractionwasuntrans-formedDMSP.
IncreasingsalinityresultedinafasterandgreateroveralluptakeofDMSPafter6h(Fig.
4),andcausedthebacteriatoaccumulateadditionalDMSPandkeepitintactintheircells.
ThefactthatbacteriasubjectedtosalinitystressaccumulateDMSP(Fig.
4)andthatDMSPalleviatessaltinhibi-tionofbacterialproduction(Figs.
1&2)representsstrongevidencethatDMSPisusedasanosmolytebynaturalheterotrophicprokaryotes.
TheintracellularDMSPconcentrationaccumu-latedundersaltadditionwashighenough(13to54mM)toprovidebacterialcellswithsomelevelofosmoprotection(Table1).
Duringsimilarexperi-mentsdonewithGBT,Kiene&HoffmannWilliams(1998)alsofoundincreasingretentionofGBTinthecellsasthesalinityincreased,withintracellularGBTconcentrationsof16,28and43mMfor7,14and25pptsalinitytreatments,respectively,incloseagreementwithourintracellularDMSPconcentra-tions.
Ourestimationoftheintracellularconcentra-tionofDMSPassumesthat100%ofcellscountedbyflowcytometrywereaccumulatingDMSP,whichisunlikelytohappeninanaturalmicrobialassem-blage.
Amorereasonableassumptionmightbethatca.
50%ofthemicrobialcellsinthenaturalsystemareactuallytakingupDMSP(seebelow),inwhichcasetheintracellularconcentrationestimatedinTable1wouldbedoubled,andcouldreach109mMforthe+10ppttreatment.
Also,inthepossibleeventofshrinkageofthecytoplasmicvolumeduetotheincreaseinexternalosmolarity(Csonka1989andreferencestherein),theintracellularDMSPconcentrationwouldbeevenhigher.
Forexample,ifthehyperosmoticshockcausedthebacterialbiovol-umetodecreaseto0.
04m3fromtheassumedvalueof0.
07m3,theintracellularDMSPconcen-trationwouldbe95mMinthe+10ppttreatmentif100%ofthecellstookuptheDMSP,and190mMifonly50%ofthecellstookupDMSP.
Theseintra-cellularconcentrationsareintherangeproducedbyphytoplankton,whichuseDMSPasanosmolyte(Reed1983,Dickson&Kirst1986,1987,Karstenetal.
1992).
Thatnotallbacterioplanktoncellswouldpartici-pateinDMSPuptakeissupportedbyseverallinesofevidence.
Firstly,asignificantfractionofthebac-terioplanktonisnotmetabolicallyactive,andthusnotlikelytoparticipateintheuptakeofDMSP.
Basedonsingle-cellassaysusingfluorescenceinsituhybridization,itisestimatedthatabout60%ofthebacterialcellsinmarinewatersaremetaboli-callyactive,althoughthisfractionishighlyvariablebothseasonallyandspatially(Lennon&Jones2011andreferencestherein).
Secondly,severalstudieslookingatthesingle-celluptakeof35S-DMSPindif-ferentenvironmentsshowedthattypically,ca.
50%oftheheterotrophicprokaryotecommunityisabletotakeupandassimilatesulfurfromDMSP(Malm-strometal.
2004,Vilaetal.
2004,Motard-Ctéetal.
2012).
Thisnumbershould,however,beconsideredasamaximumestimate,sincetheselongincuba-tions(ca.
24h)canallowfortheconversionof35S-DMSPto35S-MeSH,whichcouldthenbeassimi-latedbyorganismslackingthecapacitytoimportDMSP.
Additionally,theuptakeofDMSPrequiresspecificmembranetransporters,whosedistributionamongmarinebacterioplanktonisnotwellknown,andmightnotbefoundinalltaxa.
TransportersimportingDMSPinthecellsbelongto2superfami-lies:theATP-bindingcassette(ABC)-type(Kempf&Bremer1998)andthebetaine-choline-carnitinetransporter(BCCT)-type(Toddetal.
2010,Ziegleretal.
2010).
TheABCtransportsystemsOpuAandOpuCidentifiedinBacillussubtilisaresimilartotheProPandProUsystemsinEscherichiacoli(Boncom-pagnietal.
2000,Sleatoretal.
2001).
OpuAhasonlyalowaffinityforDMSP(inhibitionconstant,Ki=912M),whereasOpuChasahighaffinityforDMSP(Ki=39M)aswellasseveralofitsderiva-tives(Broyetal.
2015).
OneBCCT-typetransportermediatingDMSPimportiscalledOpuDinB.
sub-tilis,similartoBetPinCorynebacteriumglutamicumandCaiTinE.
coli.
Recently,anotherBCCT-typetransporteridentifiedfromMarinomonasMWYL1,DddT,hasbeenshowntomediateuptakeofDMSP.
GenessimilartodddTwerealsofoundinthe2mar-inespeciesHalomonasHTNK1andSagittulastel-lataE37(Toddetal.
2007,2010,Johnstonetal.
2008)and2isolatesfromthegutofAtlanticherring,PseudomonasandPsychrobacter(Cursonetal.
2010).
Interestingly,thegenesfortheDddTuptakesystemareoftenfoundclusteredwithotherdddgenesinvolvedinDMSPdegradation.
Onlythese4transportsystems(OpuA,OpuC,OpuDandDddT)andtheirhomologsareknowntomediateuptakeofDMSPincells.
Abetterknowledgeoftheirdistribu-tionamongthemarinebacterioplanktontaxawouldimproveourunderstandingofthepotentialglobalimportanceoftheDMSPretentionmechanismanditseffectontheglobalcyclingofDMSP.
Motard-Cté&Kiene:DMSPasosmoprotectantforbacteria143DMSPprovidesosmoprotectionatlowambientconcentrationDMSPprovidedsignificantosmoprotectionat2.
5nM(Fig.
5),aconcentrationwithintherangethatistypicallymeasuredindifferentenvironments(Kiene&Slezak2006).
MoreDMSPprovidedmoreprotection,andinhibitionofBPbyacute+10pptsalinityincreasewasalmostcompletelyrelievedbya12nMDMSPaddition,aconcentrationthatcouldbefoundduringbloomconditionsorinmicroenviron-mentsaroundleakingcells(Mitchelletal.
1985,Wolfe2000).
Ourresults(e.
g.
Fig.
6)suggestthatretentionofDMSPinnaturalsystemscouldbeenhancedinoceanicorestuarinewaters,wheresalinityishighorsubjecttostrongandrapidfluctuations.
RetentioncouldalsobepromotedathigherenvironmentalDMSPdlevels,whicharelikelytohappeninrelativelyproductiveenvironmentsandwhereintensegrazingactivitymightreleasemoreDMSPd(Archeretal.
2002,Simóetal.
2002).
However,theseresultsalsoshowthatsomeDMSPisretainedinthemicrobialcellsevenatverylowambientDMSPdlevels(0.
5nMinthisstudy;Fig.
6).
Thus,DMSPaccumulationislikelytooccurinlessproductiveenvironmentsaswell.
Asmentionedabove,DMSPretentioninbacter-ialcellshasbeenobservedinopenoceansampleswithpresumablystablesalinityandlowsteadystateDMSPdconcentrations(1.
7nM)(Kiene&Linn2000a).
Inourexperimentswithpre-incubatedfiltratescontainingonlybacteria,thereseemedtobeathresholdatwhichtheavailabilityofDMSPwashighenoughthatthecellscouldaffordtokeepsomeofitintactforacertaintime.
ForDMSPadditionsof2.
5nMandbelow(Fig.
6),theamountofDMSPincellswasthesameatambientand+10pptsalinity.
Withtheseloweradditions,mostoftheDMSPisprobablydegradedandusedtomeetbacterialenergy,carbonandsulfurdemands,leavinglittleexcessDMSPtobeaccumulated.
Thisthreshold,orinotherwordsthelevelofDMSPrequiredtoachieveasignificantaccumulationandosmoprotectiveeffect,probablydependsonthebacterialactivitylevel(hencethebacterialdemands),andisthusexpectedtobelowerinlessproductiveenvironments.
Addi-tionalstudieswillbeneededtotestthishypothesis.
DecreasedproductionofMeSHundersalinitystressExperimentalincreasesinsalinityresultedinalowernetproductionofMeSH,suggestingadown-regulationofthedemethylation/demethiolationpath-way,whilealargerfractionoftheDMSPtakenupwasretainedandaccumulatedinthecellsforosmo-protection.
Consistentwithourresults,Kiene&Hoff-mannWilliams(1998)alsosawadecreasein14CO2productionalongwithahigheraccumulationof14C-GBTinthecellsathighersalinity,indicatingare-tardeddegradationofGBT.
Salgadoetal.
(2014)reportedadifferentpatternwithculturesofRuegeriapomeroyi,wheresalinityincreasesresultedinlowerDMSbutenhancedMeSHproductions.
However,theirDMSPamendmentswere50and500M,whichis3and4ordersofmagnitudehigherthanwhatwasaddedinthisstudy,andcouldexplainthedifferentresponseobserved.
SignificanceandimplicationsofbacterialretentionfortheDMSPcycleRetentionofuntransformedDMSPinprokaryotecellsforosmoticpurposesisverylikelytohappenundernaturalconditions.
PreviousstudiesshowingthisphenomenonaddedhighlevelsofDMSPtodensebacterialcultures(Wolfe1996),whichinmostcaseswereentericbacteriathatdonotnaturallyencounterDMSPintheirenvironment(Mason&Blunden1989,Diazetal.
1992,Cosqueretal.
1999).
Here,weshowedthatthenaturalbacterialassemblagefromGF/F-filteredcoastalseawaterretainedasignificantfractionofDMSPdintheircells,evenatextremelylowambientDMSPdconcentrations(0.
5nM;Fig.
5).
Thismicrobially-retainedpoolincreasedwithboththeavailabilityofDMSPdandthelevelofosmoticstress,anddidprovideosmoprotectiontothecellsatDMSPdconcentrationaslowas2.
5nM,whichisintherangeofconcentrationoccurringinmostmarineenvironments.
Whileourexperimentsfocusedonsalinitystress,itremainspossiblethatDMSPreten-tioncouldbeprotectiveagainstotherstressesexperi-encedbybacteria,suchasoxidativestress,nutrientstarvation,andthermalshock.
TounderstandtheimpactsofthisaccumulationmechanismontheDMSPcyclinginmarinewaters,wefirstneedtoestablishwhetherthispoolcanrepre-sentasignificantfractionofthetotalanddissolvedDMSPpools.
Fig.
5showsstrongrelationshipsbe-tweentheaccumulationofintracellularDMSPandtheexternalDMSPconcentration,atambientandin-creasedsalinity.
Under+10pptsalinity,thisaccumu-lationwaslinear,and54%oftheDMSPdinitiallypro-videdwasfoundinthecellsafter1.
5h.
TheambientsalinitytreatmentfollowedthesametrendatlowerDMSPdlevels.
WhileretentioncontinuedtoincreaseAquatMicrobEcol76:133–147,2015144above3.
5nMaddedDMSPdintheambientsalinitytreatment,thefractionofaddedDMSPdaccumulateddecreasedabove3.
5nM,andwasca.
33%oftheDMSPadditionatthe12nMlevel.
Themicrobiallyretainedpoolthusrangedfrom33to54%oftheextra-cellularpoolofdissolvedDMSP,whichishigherthanpreviousestimates(10to15%oftheDMSPdpool)fromwholeseawater(Fig.
1)andin1doldfiltratein-cubations(10to37%;Lietal.
2015).
Lowlevelsofla-bileorganicnutrientsinour3to4doldGF/Ffiltratelikelyresultedinalowbacterialgrowthrate(Mc-Manusetal.
2004).
AfastergrowingcommunitywouldprobablydegradeDMSPfaster,leadingtoalowerretentionofuntransformedDMSPinthecells.
Nevertheless,themicrobially-retainedpoolofDMSPcanbeasignificantfractionoftheoperationally-definedDMSPdpool,andcouldimpairthemeasure-mentoftrulydissolvedDMSPsincethetraditionalmethodsforDMSPdinvolvefiltrationthroughGF/F-typefilters,whichletsomebacteriapassthrough(Leeetal.
1995).
Therefore,theoperationally-definedDM-SPdwilloverestimatethe'dissolved'DMSPpoolifthesebacteriahaveaccumulatedDMSPintheircells(seealsoLietal.
2015).
SuchanoverestimationcouldalsoindirectlyaffecttheDMSPturnoverratecalcula-tionsthatarebasedontheDMSPdconcentration.
WhetherbacteriasignificantlyaffecttheturnoverofDMSPbytakingupandretainingDMSPwithintheircellsisanotherimportantquestion.
Witharesi-dencetimeofdays,themicrobially-retainedpoolap-pearstoturnovermuchmoreslowlythantheextra-cellularDMSPpool,slowingdowntheoverallDMSPcyclingandthefluxesofsulfurandcarbonwithinthemicrobialfoodweb.
AccumulationofDMSPinbacte-rialcellscouldalsoenhancethetransferofDMSP-sulfurtohighertrophiclevelsthroughbacterivory.
ThefateofingesteduntransformedDMSPviagraz-ingisstillpoorlyunderstood.
Moststudiesaddress-ingthisquestionhavefocusedontheassimilationofphytoplankton-DMSPbyherbivores.
Salóetal.
(2009)foundthat32%oftheDMSP-sulfuringestedbythedinoflagellateOxyrrhismarinapreyingonthediatomThalassiosirapseudonanawasassimilatedintobiomass,whilethebiggestfractionwaseitherconvertedtoDMSorreleasedasDMSPdtotheextra-cellularenvironment.
Innaturalconditionshowever,grazersmightbeeatenbypredatorsbeforedigestingalltheDMSP,passingafractionofituptohighertrophiclevels(Tang&Simó2003).
NostudysofarhaslookedatthefateofDMSPingestedbyprotists(ciliatesandflagellates)feedingonbacteria.
Itisgen-erallyassumedthatthemajorimpactofmicrozoo-planktongrazingontheDMSPcycleistoenhancetheoverallproductionofDMS(Wolfe&Steinke1996)bydesegregatingtheDMSPandDMSP-lyaseduringthedigestiveprocess,andthusreducingtheoverallassimilationofDMSP-sulfurbythetrophicweb.
ThisDMSproductionviagrazingcouldbefur-therincreasedifDMSPretentionbybacteriasome-howmakesthemmoreappealingtomicrograzers,forexamplebyallowingthemtoattaingreatersize(viagreaterosmoticvolume).
Overall,themajorimpactofaccumulationandretentionofDMSPintobacterialcellsislikelytobeageneralslowingdownoftheturnover,andpotentiallyahighertrophictransfertograzers.
Ontheotherhand,theretentionofDMSPprovidesbacteriawithaquickandefficientprotec-tionagainstosmoticstresses,whichallowsthemtoprocessorganicmatterandrecyclenutrientsunderawiderrangeofosmoticconditions,maintaininghealthyecosystemfunctioning.
CONCLUSIONSDMSPiswellrecognizedasamajorsourceofre-ducedsulfurandcarbonforbacteria.
Itismostoftenregardedasanextremelylabilecompoundinthedis-solvedorganicmatterpool,withafastturnovertime.
OurresultssuggestanosmoprotectiveroleforDMSPinmarinebacterioplankton,expandingtheroleofmarinebacteriainDMSPcyclingbeyondtheirabilitytodegradeDMSP.
WehaveshownthatDMSPisaccu-mulatedinthecellsandusedasanosmolytebypro-karyotesinanaturalmarinesystem,relievingacuteosmoticstressevenatlowDMSPdconcentrationstyp-icallyfoundinthesenaturalsystems.
DMSPcanberetainedinbacterialcellsforseveraldays,leadingtoaslowturnoverofthispoolcomparedtoextracellularlabileDMSP.
EnhancedretentionofDMSPinbacteriaundersalinitystressappearstobedue,inpart,totheinhibitionofthedemethylation/demethiolationdegra-dationpathway.
BydivertingDMSPfromfasterde-gradationpathways,thismicrobially-retainedpool,whichcanrepresentasignificantfractionoftheoper-ationally-definedDMSPdstandingstock,canslowdowntheoverallcyclingofDMSPandallowtrophictransferofDMSPtobacterialgrazers.
Acknowledgements.
WethankA.
Ortmann(UniversityofSouthAlabama/DISL)forherhelpwiththeflowcytometrytechnique.
SupportforthisresearchwasprovidedbygrantsfromtheNSF(OCE-1342699,OCE-1436576)toR.
P.
K.
J.
M.
-C.
wassupportedbytheUniversityofSouthAlabamaandfel-lowshipsfromtheDauphinIslandSeaLabandtheFondsquébécoisdelarecherchesurlanatureetlestechnologies(FQRNT).
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EnvironMicrobiol3:304311Editorialresponsibility:JosepGasol,Barcelona,SpainSubmitted:June17,2015;Accepted:August19,2015Proofsreceivedfromauthor(s):October18,2015