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CharacterizationoftheiridescenceandmotilitymutantsinTenacibaculumsp.strainECSMB88.NadiaHerreraCaliforniaInstituteofTechnology2017MicrobialDiversityCourseMiniprojectreportIntroduction

SincetheestablishmentoftheTenacibaculumgenuswiththediscoveryoftwodistinctspeciesin2001,thenumberofspeciesaddedtothegenushasgreatlyincreasedto23individualspecies(1).ThedistinctTenacibaculumspecieshavebeenreportedasisolatesfrommanypartsoftheworld—fromChinatoPeru--aspartsofmarineorganisms,rockyshores,andrecently,deepseasediments(2-7).Tenacibaculumwasinitiallyidentifiedasoriginatingfrommarinesponges,butthegenuswasquicklypopulatedbyspecieswhichwerebeingisolatedfromfishpathogens(3,4,6,7).Itwasnotuntilmorerecentlythatagroupstartedidentifyinga“glitter-like”iridescenceintheTenacibaculumgenus,asoriginatedbystudiesonCellulophagalytica,whichlaterdevelopedmeansforenrichmentforiridescentTenacibaculumisolates(2,8-10).

Iridescence,however,hadnotgoneunnoticedandhadbeendescribedintheliteratureinreports,asearlyas1950(11).Achallengeinpresentingiridescenceinstancesinabacterialisolatewasaccompaniedbythelackofcolorphotographywhichcouldbeusedinthosereports.Thisresultedinvariedmeansbywhichauthorsreportedanddescribedthecharacteristicofiridescence,andassuch,ithasremainedarelativelyunderstudiedfacetofmicrobiologicalphysiology.Interestingly,asidefromiridescence,bacteriawhichhavethisphenotypealsoutilizeglidingmotility.Glidingmotilityhasbeenmuchlessstudiedthanflagellarmotility,andassuchremainsanotherunderstudiedfacetofmicrobialphysiology(12).Inrecentliterature,bothglidingandiridescencehavebeenstudiedinCellulophaga,Tenacibaculum,andAquimarina(8).

AniridescentTenacibaculumisolate,correspondingtoTenacibaculumsp.strainECSMB88,hasbeenisolatedbyastudentinthe2017cohortofMicrobialDiversity,MelissaKardishfromtheMarineBiologicalLaboratory’sStonyBeachinWoodsHole,Massachusetts.Intriguinglyenough,ECSMB88waspreviouslyisolatedfromtheEastChinaSeaBiofilmbyYang,J.L.,andGuo,X.P.intheFisheriesandLifeScienceinstitutioninChina,asstatedintheirdeposition,KU982641,intheGenBankdatabase.ThisiridescentTenacibaculumisolatereadilygrowsglidingcolonieswhichshowiridescenceuponthefirst12hoursofincubationat30°C.Assuch,thisparticularisolatepresentsamodelorganisminwhichtostudygenesbehindglidingandiridescenceusingobservationsonwholecolonies.Thisreportpresents7mutantstowhichdefectsinglidingmotilityandiridescencewereintroducedbyuseofachemicalmutagenesis

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screenusingMethylMethanesulfonatetomodifytheDNAinTenacibaculumsp.StrainECSMB88.--MaterialsandmethodsIsolationofTenacibaculumsp.StrainECSMB88 SamplesforisolationwerecollectedbyMelissaKardishfromStonyBeachinWoodsHole,Massachusettsat41.529°E,70.674°N,at8A.M.duringthelowtide.Aseagrassrootwascollectedfromtheeastendofthebeach,nexttothejetty.Aftercollection,theseagrassrootwasstoredinseawateruntilitwastransportedtothelab.Onceinthelab,thesamplewassubjectedtothroughmixingusingthevortex.TheresultingsupernatantwasseriallydilutedandplatedontoSeawatercompletemediumplatesandincubatedat30°C.Thesequenceofthesamplewasfoundbydoing16sPCRwithprimers8Fand1391R,asdescribedbefore(13).ThePCRproductwassubmittedforsequencingandtheresultwasalignedusingNCBIBLASTn.Aphylogenytreecomparingtherelationofthetop10hitsof16sRNAsequencingresultwasmadeusingaMUSCLEalignmentfollowedbyuseofMEGA7withdefaultparameterstogenerateatreebasedonmaximumlikelihoodbasedonpreviouswork(14,15).MMSMutagenesis Methylmethanesulfonate(MMS)wasacquiredfromSigma-Aldrich,andusedatafinalconcentrationof40uMinallreaction.10uLofaliquidovernightcultureofTenacibaculumsp.StrainECSMB88wasusedtoinoculatea5mLcultureinSeawatercompletemediumandwasgrownforaround7hoursoruntilitreachedtoanOD600nm=0.5-0.8,soastoobtaintheorganisminexponentialphase.Onceexponentialphasewasreached,a1mLaliquotofcellswasremovedfromthecultureandsubjectedtoafinalconcentrationof40uMMMS(4.44uLof99%MMSinto1mLofcells)foratotalof5minutesat30°C.Thecompletedreactionwascentrifugedusingatabletopcentrifugeoperatingat12,000RPMfor2minutes.Supernatantwasdiscardedandtheremainingcolonywasre-suspendedwithseawatercompletemedium.There-suspensionwasseriallydilutedupto10-8.Atotalof80plateswereusedforspreadingthecells,todistributethenumberofcellsattainedandscreeneverydilutionfortheemergenceofindividualcolonies.Todistributethecellsspread,100uLofinoculumwasspreadineachof20platesperdilution:10-5,10-6,10-7,and10-8.AnadditionalseawatercompletemediumplatewasinoculatedwithwildtypeTenacibaculumsp.StrainECSMB88,soastoanalyzewildtypecolonies.Aftera2-dayincubationperiodatroomtemperature(~20°C)colonieswereanalyzedandpickedforfurtheranalysis.Mutantselectionandplatinganalysis Initially,colonieswerepickedonthebasisofthreevisiblephenotypes,lossoriridescence,lossofmotility,orgainofmotility.Inthelattercase,thecolonywouldappearasmorediffusethanawildtypestandard,whereaslossofmotilitywouldbemanifestedinacolonywhichlosttheirregularedgesandformedalessmotileandcompactarrangement.Lossofiridescencewouldbeanalyzedbyvisuallyinspectingcolonieswhiletiltingtheplatetoallow

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lighttoshinethroughthecoloniesunderinspection.Fromtheinitialscreen,34colonieswerepicked,andstreakedforisolationtoverifythephenotype.Onceverified,6mutantcolonieswereusedforsubsequentanalysisonseawatercompletemediumsupplementedwith1%papermateink.Eachcolonywasplatedbypipetting2-5uLofeitherre-suspendedcolonyinseawatercompletemedium,orliquidculture.Theresilienceofthemutationwasfurtherobservedbyplatingthecellson½dilutedseawatercompletemediumplateswith1%,1.5%(sameasregularplates),2%,and2.5%agarconcentrations.Stereomicroscopy StereomicroscopyimagesofindividualcoloniesonplatesweretakenusingaZeissSteREODiscoveryV.8microscopesupplementedwithanexternallightboxforangledilluminationoftheiridescentcolonies.Spectroscopy

Asalightsource,weusedanOSRAMM11Halogenlightbulb8V/20W.ThelightenergywasdirectedinaZEISSmobilefiberandfocusedbyamicroscopecondenserlenswithanapertureof1.25underanangleof38°ontothehorizontallypositionedcolonyonaplate.Iridescentlightwascollectedintoaglassfibermountedatadistanceof1cmverticallyabovetheobjectandconnectedtoaSpectralEvolutionSR-1900SpectroradiometerSN15782C0.SpectrawererecordedandanalyzedbytheDARWINsoftwareofSpectralEvolution(1CanalStreet,UnitB1,Lawrence,MA01840).Lightmicroscopy Liquidmountslideswerepreparedshortlybeforeimaging.LightmicroscopyimagesofindividualcellsfromliquidcultureweretakenonaZeissAxioImager2microscope,using100xmagnificationandaphasecontrastplateforeaseofvisualization.Scanningelectronmicroscopy Wholecolonieswhichhadbeengrowingfor12hoursweredehydratedforscanningelectronmicroscopy(SEM)studiesutilizinganosmiumtetraoxideandglutaraldehydefixationmethod,followedbydehydrationwithethanolandhexamethyldisilazane(HMDS).Dehydratedcolonieswerethenmountedontoaspecimenstubusingdoublesidedcarbontape.A10nmcoatofplatinumwasappliedusingaLeicasputtercoater.Foradetailedprotocol,pleaseseetheendofthisreport.ImagingofthesampleswasdoneonaSUPRA™40VPFieldEmissionSEM,equippedwithGEMENIcolumntechnologysupplementedwiththeThermoNoranSystemSIXEDS.

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ResultsPhylogenytreebasedon16SrRNAsequencing

Thephylogenytree(Figure1)indicatedthatourisolatedhasacloserelationwithTenacibaculumspstrainECSMB88,andTenacibaculummesophiliumstrainHMG1.Duetothesimilarityoftheseresults,wehaveoptedforusingthenameofthefirsthitintheentiretyofthisreport.Assuch,theisolatedstrainisreferredtoasTenacibaculumspstrainECSMB88.

Figure1-Phylogenytreecalculatedfromthetoptenhitsof16srRNAsequencingofourisolate.Thephylogenytreedisplaysacomparisonof10hitsfromtheNCBIBLASTnresultsof16srRNAsequencingdataobtainedfromourisolate.Thescalebarindicatesthebranchdistancescale.

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Firstroundofmutantsselected Thefirstseriesofmutantswereselectedindividuallyfroman80platearray.Coloniesthatspreadoutrelativetoawild-typecomparisonwereconsideredtobegainofglidingmutantsand,andre-struckonseawatercompletemedium,asshowninblueboxesinfigure2.Ontheotherhand,colonieswhichappearedaslossofglidingmutantshadveryroundcolonieslackinganirregularedge,thesecolonieswerepickedandareshowninorangeboxesinfigure2.Finally,colonieswhichappearedtoloseiridescenceorchangeiridescencefromthewildtypegreen/yellowhuewerepickedforfurtheranalysis,andareshownasyellowboxesinfigure2.

Figure2-Stereomicroscopyimagesofmutantsselectedforanalysisbyre-streakingofasinglemutantcolony.Instancesofgainofgliding,lossofgliding,andlossofiridescencearehighlightedinblue,orange,andgoldboxes,respectively.Analysisofselectedmutantsfromfirstround Tounderstandthephysiologicalchangesbehindthedifferentphysicalobservationsatthecolonylevel,themutantsofinterestwereplatedonseawatercompletemediumsupplementedwithink.Theblackinkshowedthestrikingdifferenceiniridescenceasthecolonygrowsontotheplate,withgreeniridescencetypicallyresidinginthecenterofthecolonyandrediridescenceresidingontheedgesofthecolony(Figure3).Inourmutationsofinterest,thisiridescencewasonlychangedinmutants4and5,whichhadbeenhighlightedaslossofiridescencemutantsinthescreenillustratedinfigure2.Onceabasalphenotypewasobserved,thecolonieswereplatedonlow-nutrientmedium,½seawatercompletewiththesameamountofagarasisusedintypicalseawatercompletemediumplates,1.5%.Theseplates

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showedthatwithlowernutrients,theedgeoftheglidingcolonieswasspreadwidelywhilemaintainingacorrugatedshape,asseeinwildtype,andmutants1,2,4,and5(Figure3).

Ontheotherhand,mutantswhichhadbeenidentifyingashavingalossofglidingmotilitywerespreadout,buthadevenlycircularedgesasseeninmutants3and6(Figure3).Inaddition,itwasclearthatcolonies3and5hadstrikinglydifferentorganizationthantherest,highlightingacircularcolonysurroundedbyathinnerdefinedringandtaperedwithathinnerlayerofiridescentcells.Thisphenotypewasmuchreducedwhenanalyzingthesamecoloniesin½seawatercompleteand1%and2.5%agar(figures5-6).Astrikingobservationwasthatin½seawatercompletemediumwith1%agar,mostofthecoloniesspreadthoroughly,andevendisplayedlossofiridescenceinmutants2and5(figure5).Intriguingly,mutant3didnotspreadthoroughly,butinsteadremainedinasmallandcircularcolonyshape(figure5).Lastly,uponincreasingtheagarconcentration.In½seawatercompletemediumto2.5%,iridescencewasverydistinctfromtheotherconditions,andagreenhuewasobservedontheoutsideofwildtype,andmutants1-3,and5-6(Figure6).Ontheotherhand,themutantwhichhadbeenselectedforalossofiridescence,mutant4,waspigmentedcompletelydifferentlyanddisplayedverylittleiridescenceamidstaredhue(figure5).Collectively,theeffectofagarconcentrationandnutrientavailabilitygreatlyimpactshowthemutationsmanifestthemselves.Thisindicatesthatitiscriticalto“makenicewithyourbug”andplateitonmultiplemediainthepursuitofinvestigatingtheeffectsofamutation.

Figure3-Colonycomparisoninseawatercompletemedium,using1.5%agar.Wildtypeisdisplayedontheleft-handpanel,whereastheselectedmutantsareontheright-handpanel.Iridescentpropertiesarehighlightedbytheuseofblackinkontheplates,demonstratingthevariabilityofcolorsindifferentregionsofthecolony.

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Figure4-Colonycomparisoninseawatercomplete,using½concertationofmediumnutrientswhilemaintainingtherigidityoftheagarfromregularseawatercompleteplates.Theseplateshighlightadditionalglidinginallofthemutantsanalyzed,withdefinedchangesincolonymorphologyinmutants3and6.

Figure5-Colonycomparisoninseawatercomplete,using½concentrationofnutrientsinmediumand1%agar.Inthismedium,colonieswhichwerecharacterizedasgainofglidingweremuchspread,whereascolony3wasfurtherestablishedasalossofmotilitycolony.Theiridescenthueinthecoloniesbecomesdiminishedinwildtype,andmutants1-2,4-5.

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Figure6-Colonycomparisoninseawatercomplete,using½concentrationofnutrientsinmediumand2.5%agar.Inthismedium,ahardersurfacepreventsmostcoloniesfromglidingfar.Inaddition,iridescenceismanifestedasagreenpigmentalongtheedgesofmostcolonies,whereasthelossofiridescenceisprominentinmutant4.Analysisofcellmorphologyinindividualcolonies Lightmicroscopyoncellsobtainedfromliquidcultureofvaryingmutantsshowedthattheyallcontainedamixedpopulationofrodswithvaryinglengths,ashighlightedinFigure7.Inthisanalysis,itwasevidentthatamixedpopulationofrodswashomogeneousamidstallcells,withwildtype,andmutants2,4and5displayingbothaninstanceofalongrodandaformationofcellularorganizationonaglassslide.Interestingly,mutants1-3,and6onlysharedoneofthosecharacteristicswithwildtype.Sincecellularorganizationappearstobeveryvariedamidstthemutants,itwasimperativetoanalyzetheirorganizationatthewholecolonylevel,soastounderstandcellularorganizationatthecolonylevel.

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Figure7-Lightmicroscopyimagesofthemutantsselectedforanalysis.Elongatedcellshapesarehighlightedusingredarrows,whereaseventsthatappeartobeearlycellularorganizationarehighlightedusingyellowarrows.Ingeneral,mostmutantsshareeitheroneorbothofthesecharacteristicswithwildtype.SEManalysisofcoloniesandmutants SEManalysisofourcoloniesallowedfortheinvestigationofsubcellularorganizationintheentiretyofacolony.InpreparationforSEM,itwasnotedthattheiridescentpropertiesofthecoloniesappearedtosurviveddehydration,asshowninfigure8.OnceanalyzedwithSEM,immediatecolonyorganizationwasnotevidentinanyportionofthecolonyforanyofthemutationsobserved.Ahomogeneousmixtureofcellsizeincolonyformationwasobservedinwildtypeandmutants1,2,4-6(Figure9).Thisresultlikelyexplainstheglitter-likeiridescenceobservedincoloniesonagar,astheiridescenceislikelytheresultofasmallgroupofcoloniesforminganorderedarrayamidstanarrayofcellsinagivencolony.Interestingly,ourlossofiridescencemutantdisplayedanorderedarrayofcellswhichseemedtohaveaperiodicstructurewithinindividualclustersandwithincellsthemselves(Figure9).Thisresultisindicativeofastructuraldifferencecorrelatedwithachangeofpigmentinacolony.Ontheotherhand,thecellsinmutant3appeartoreleaseamaterialwhichislikelyexopolysaccharideusedinbiofilmformation.Characterizationofiridescentpropertiesinthesemutants,isthereforeofinterestforfurtherdifferentiation.

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Figure8-SEMcolonydehydrationafterfixation.Huesofpinkandgreenonthecolonysurfaceappeartomanifestaretentionofcolonyiridescencethroughtheprocedureofdehydration.Colonysizewasreducedtoabout1/10oftheoriginalsizeandcoloniesbecamecorrugatedfromdehydrationafterpreparation.

Figure9-SEMofcolonies.SEMonindividualcoloniesshowedamesh-likestructureofcellsofvaryingsizesonthecolony,regardlessoflocationofimagingonthecolony.Mutant3showedaparticularlydensebedofextracellularmaterialaccumulationwhichislikelyexopolysaccharides.SpectroscopiccharacterizationofmutantsSpectroscopystudiesonindividualcolonies Spectroscopymeasurementsweretakenonindividualcoloniesgrownonseawatercompleteplatesinblackagar.Theseplatesprovidedabaseforreflectionmeasurementsoffoftheiridescenceofthecolony.Weobservedthatwildtypehasstrongestreflectionsat540nm,whichisrightinthegreenzoneofthelightspectrumandinaccordancewiththecolorthatweseebyeye.Thispeakisshiftedinmostmutants,butthemoststrikingchangeisinmutant4,

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wherethepeakmovestothe582nmrange,whichisclosertotheyellowspectrumanddifferentiatesthiscolonyashavingdifferentspectroscopicreflections.Ourresultssuggestthatthisprocedureofmeasuringiridescenceisawaytoprovidequantificationofdifferencesobservediniridescence,andcouldbeawaytotrulydiscernbetweendifferentiridescencetypes.

Figure10-Spectroscopyonindividualcoloniesgrownonseawatercompleteagarplatessupplementedwithink.Discussion

Wehaveisolatedcoloniesthathavedefectsinglidingabilityandiridescentcolor.Studyingthegeneticbasisbehindthiswillshedlightontwofacetsofbacterialphysiologywhicharenotwellunderstoodtodate.Thoughitisinitiallyobservedthatcoloniesformlittleorder,theyappeartoretainiridescenceafterdesiccation.Inaddition,theiriridescentpropertiesseemtoremainunchangedafterdehydration.Inordertofurtheranalyzecolonyorganizationatthecellularlevel,itwouldbenecessarytoimageagrowingcolonyusinganinvertedmicroscopesetup,orstudyingawholecolonyusingtransmissionelectronmicroscopy,whichcouldshedlightoncellularorganizationattheultrastructurallevel.

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Thisorganismhasalotofinterestingfeatureswhicharetraceable,andmanycharacteristicsandpropertieslefttoexplore.Forinstance,throughoutthistime,itisdifficulttousecentrifugationat3400RPMtopelleta50mLmassofcells,evenafterextendedspinning.Itislikelythatthesecellsarebeingkeptinsuspensionbytheirproductionofexopolysaccharideinthegrowthmediumoncetheyreachlagphase,asthisisnotobservedincellsgrowntoexponentialphase.Infact,studieshavebeendonewhereafoambeadisseenglidingalongacellinliquidmedium,soastosuggestthattheirglidingmotilityisactivewhilegrowingonliquidmedium,despitethelackofasurfacetoglideon(16).

Inordertobetter-understandthesephenotypicmutations,itessentialtosequencethegenomeofthecoloniesofinterest,soastostudythegeneswhicharebeingmutatedtocreatetheeffectsseen.Outofthemutantspresentedinthisstudy,therearethreedistinctcoloniesthatshouldbestudiedtostarttounderstandpatternsofchange,andthosearewildtype,alossofmotilitymutant3,andalossofiridescentmutant5(Figure11).Onceweareabletomapthemutationstothegenome,complementationstudieswillallowustoestablishthegenesbehindthemutationsobserved.Fortunately,inthelastyearageneticsystemhasbeenestablishedforasimilartypeofbacteriawhichcanbeusedforcomplementationstudies(17).Ultimately,theseconstructscanprovetobethebeginningofaninvestigationtowardstwobacterialphenotypeswhichhaveyettobethoroughlyunderstood.

Figure11-Wholegenomesequencecandidates,colonyandSEMimages.Amidstallofthemutationsinthisstudy,thesethreecandidateswouldallowforinitialunderstandingofthemachinerybehindmotility.

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andtaxonomicstudyofmarineCytophaga-likebacteria:proposalforTenacibaculumgen.nov.withTenacibaculummaritimumcomb.nov.andTenacibaculumovolyticumcomb.nov.,anddescriptionofTenacibaculummesophilumsp.nov.andTenacibaculumamylolyticumsp.nov.IntJSystEvolMicrobiol51,1639-1652

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SupplementalinformationProtocolforSEMdehydrationandfixationofabacterialcolony

1- Usingarazorblade,cutoutthecolonyfromanagarplate.2- Scoopthecolonyontothelidofa1cmdiameterpetridish3- Add500uLoffixativeagent,Osmiumtetraoxideinwaterat1%concentrationtothe

bottomofthepetridishandspreadthroughoutplateevenly.4- Carefullyplacethecolonyovertheliquidinthebottomoftheplate,andadd300uLof

additional1%osmiumtetraoxidesoastoformameniscusaroundthecolony.Donotre-suspendcolony.

5- Allowfixationtoproceedfor1houratroomtemperature,coveryourdish.6- Pipetteoutthefixative,andreplacewith800uLof3.5%glutaraldehydein0.1M

cacodylatebuffer.7- Allowfixationtoproceedfor2hoursatroomtemperature,coveryourdish.8- Pipetteoutthefixativeandreplacewith800uLof0.1MCacodylatebuffer.9- Allowwashingtoproceedfor15minutes,andrepeatstep8foranadditional15

minutes.10- Atthispoint,yourcolonyshouldbedarkened.Pipetteoutwashingsolutionandadd800

uLof1%Osmiumtetraoxidein0.1Mcacodyltebuffer.11- Allfixationtoproceedforanhouratroomtemperature,coveryourdish.12- Removesupernatantandadd800uLofdistilledwater.13- Allowwashingtoproceedfor30minutes,andrepeatstep12foranadditional30

minutes.14- Todryyoursample,pipettegently,useanelectricpipettethatwillallowfordispensing

atlowspeed.Pipetteoutthewaterandreplacewith4mLof50%ethanol.15- Allowdryingtoproceedfor15minutes.16- Pipetteoutethanol,andreplacewith4mLof70%ethanol.17- Allowdryingtoproceedfor15minutes.18- Repeatsteps16-17with80%,90%,95%,and100%ethanol(twicefor100%).19- Afterfinalstep,removeethanolandreplacewith4mLofa1:2solutionof

Hexamethyldisilazane(HMDS):Ethanol.20- Allowdryingtocontinuefor20minutes.21- PipetteoutfirstsolutionofHMDS:Ethanolandreplacewith4mLofa2:1HMDS:Ethanol

solution.

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22- Alldryingtooccurfor20minutes.23- Repeatspets21and22with100%HMDS.24- Onceyoureachyourfinalstepof100%HMDS,pipetteoutliquidcarefully,andallow

colonytodryslowlybyleavingthecoveronthepetridishovernight.Oncedrythecolonieswillbebrittleandmuchreducedinsize.