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Siderophore

By Curtis Tucker,2014-07-24 02:35
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SiderophoreSidero

    Siderophore

Jan.2008,Volume2,No.1(SerialNo.2)JournalofLifeSciences,ISSN1934-7391,USA

    ;Siderophoreproductionfrom27filamentousfungalstrainsandanovel ;siderophorewithpotentialbiocontrolapplications ;fromAspergillusnigerAn76

    ;SUNHongZHANGWeiCOt1,LUXuemei,GAOPei-ji

    ;rStateKeyLaboratoryofMicrobialTechnology,ShandongUniversity,Jinan25010&amp

    ;ChinaJ

    ;Abstract:Among27filamentousfungi,astrainAspergillus ;nigerAn76gavethehighestsiderophoreyieldevenwhen ;culturedonnaturalmediumorminimalmediumcontainingl ;mmolL-Fe.Whereasformostotherstrains,thecapacitiesof ;siderophoreproductionwasrepressedastheconcentrationsof ;Fe3washigherthan20pmolL.Lowerferriciron

    ;concentrationsweremoreconducivetosiderophoresynthesis ;forthosestrains.TheAn76siderophorewaspartiallypurified ;throughaseriesofchromatographystepsusingSephadexLH20 ;andCMSepharoseFastFIowcolumns.Itsstructural

    ;characteristicsdifferedfromtypicalsiderophores,suchas ;catecholatesorhydroxamates,andnoaminoacidsorpeptide ;bondsweredetected.Itmaythereforerepresentanewtypeof ;siderophorestructure.TBAassayandESRtrappingstudies ;showedthatAn76siderophorehadremarkablescavenging ;activityofhydroxylfreeradicalsinvitro.Theantibiotic ;activitiesofsiderophoresproducedbyAn76weretestedagainst ;29bacterialstrains,includingplant,animal,andhuman ;pathogens.Thegrowthof14bacterialstrainscouldbe ;completelyinhibitedatasiderophoreconcentrationof8mgml

    ;withanincubationtimeof30min.Evidencesuggeststhatthe ;An76siderophorehaspotentialvalueasanantioxidantanda ;biocontrolagentagainstpathogenicmicroorganisms. ;Keywords:antibioticactivity;antioxidant;ESR;hydroxylfree ;radicals;siderophore

    ;1.Introduction

    ;Acknowledgments:ThisworkwassupportedbytheNational ;NaturalScienceFoundationofChina(No.30470051).the ;MajorStateBasicResearchDevelopmentProgramofChina ;(973,No.2004CB7197),andtheNaturalScienceFoundation ;ofShandongProvince(No.Y2oo4DO7.

    ;GAOPei-ji(1936),male,professor,Ph.D.supervisor,

    ;nationmodelteacher;researchfields:microbialdegradationof ;thewoodfiber,foundationmicrobiologyteaching. ;LUXuemei(1968),female.Ph.D.,professor;research ;field:biOdegradationofcelluloseandlignin. ;Ironisanutritionallyessentialtraceelement.It ;hascertainimportantfunctionsinthemetabolic ;processesofaerobicorganisms,suchasphotosynthesis, ;respiration,andsynthesisofDNA.Althoughabundant ;innature,irontendstoformhighlyinsoluble ;hydroxidesintheaerobicneutralpHenvironment.The ;concentrationoffreeferricioninsolutionatbiological ;pHisprobablynotgreaterthan10I8molL[1-31.

    ;Undersuchironlimitingconditionsmicroorganisms ;synthesizeandexcreteavarietyofhighaffinity,low

    ;molecularweight(<l,000Da),ferricchelating

    ;compoundscalledsiderophores,whichspecifically ;solubilizeferricironinanextracellularaqueous

    .In ;environmentandtransportitintothecells[2-4;general,siderophorescanbedividedintothreemajor ;categoriesbasedontheirchemicalstructure: ;catecholates.carboxylatesandhydroxamatesI’.

    ;Mostfungalsiderophoresarehydroxamates,however ;zygomycctcscanproducecarboxylatcs(e.g., ;rhizoferrinproducedbyvariousMucorales)15-91. ;Siderophoreshave&tenbeensuggestedtoplayarole. ;inpathogenicvirulencebyfacilitatinggrowthunder ;ironlimitingconditions,becausetheacquisitionof ;ironisakeystepintheinfectionprocessH0-131. ;Moreover,siderophoresareknowntoplayarolein ;thebiologicalcontrolofpathogenicmicroorganisms ;bychelatingiron,therebyinhibitingtheirgrowthor ;metabolicactivity.Biosynthesisanduptakeof ;siderophoresrepresentpossibletargetsforantifungal ;l9

    ;Siderophoreproductionfrom27filamentousfungalstrainsandanovelsiderophorewithpo

    tentialbiocontrol

    ;applicationsfromAspergillusnigerAn76

    ;chemotherapy,becausehumancellsdonothavethese ;biochemicalpathways,,15].Inthisstudy.we

    ;investigatedsiderophoreproductionfrom27fungal ;strains,andtheeffectsofferricionconcentrationon ;siderophoreproduction.Anovelsiderophorewas ;partiallypurifiedfromAspergillusnigerAn76,its ;antibacterialactivitiesagainst29bacterialstrainsand

    ;scavengingeffectsofhydroxylfleeradicalswere ;studied

    ;2.Materialsandmethods

    ;2.1Bacterialandfungalstrains

    ;Atotalof27flamentousfungi,includingplant

    degradingfungi ;pathogenicfungiandlignocellulose

    ;strains,werescreenedforhighyieldingsiderophores. ;TheantibacterialactivitiesofAspergillusnigPrAn76 ;siderophoreweretestedagainst29bacterialstrains, ;includingplant,animal,andhumanpathogens(Table ;1).

    ;2.2Siderophoreproduction

    ;Threegrowthmediaweretestedtodetermine ;optimalconditionsforsiderophoreproduction,one ;minimalmediumandtwokindsofcompletemedia, ;wheatbranmediumandpotatodextrosemedium.The ;minimalmediumwascomposedasfollows(g): ;NaNO3,6;KCI,0.52;KH2PO4,0.3;MgSO4?7H20,0.5; ;glucose,15;and1mltraceelementssolution(gL,

    ;(NH4)2MoTO24?4H20,0.05;FeSO4?7H20,I; ;ZnSO4?7H20,8.8;CuSO4?5H20,0.4;MnSO4?4H20, ;0.15;Na2B4O7?10H20,0.1).Therespectivemedia ;wereinoculatedwitheachfungalstrainandincubated ;at28.Cwithshakingat170rpmfor7days.Sampling ;wasconductedevery24h,andsiderophoreproduction ;intheculturefiltrateswasdetectedbytheuniversal ;ChromeAzurolS(CAS,Sigma)assay[161.

    ;Siderophoreproductionwasindicatedbythecolor ;changeofCASsolutionfrombluetoorange, ;measuredbythedecreaseinabsorbanceat630nm. ;Allglasswarewassoakedin6molLHC!overnight ;andrinsedwithdoubledistilledwaterseveraltimesto ;20

    ;removetracesofiron.Todeterminetheinfluenceof ;irononsiderophoreproduction,different ;concentrationsofFeCl3wereaddedtotheminimal ;medium.Theferrousionconcentrationintheculture ;mediumwasmeasuredwithl,10phenanthroline

    ;spectrophotometryat510nmu71.

    ;2.3IsolationandpurificationofAn76

    ;siderophore

    ;A.nerAn76wasculturedinminimalmedium

    ;for7daysat28.C,withshakingat170rpm.Mycelia ;wereremovedbyfiltrationandthesupernatantpassed

    ;throughanultrafiltrationmembrane(NMWL:l,000). ;Theleadacetateprecipitationmethodwasusedto ;isolatesiderophores【】8_.

    ;Saturatedleadacetate

    ;solutionwasaddedtotheultrafiltrateuntilnonew ;whiteprecipitateformed.Theprecipitatewas ;collectedandwashedwithdoubledistilledwateruntil

    ;thesupematantgaveaCASnegativereaction.The

    ;precipitatewassuspendedindoubledistilledwater

    ;andaeratedwithexcesshydrogensulfidegas.The ;blacksediment,PbS,wasremovedbypaperfiltration, ;andthefiltrateconcentratedbyrotaryevaporation. ;Theconcentratedsolutionwaspurifiedbygel ;filtrationonaSephadexLH20column(Pharmacia), ;withthreebedvolumesofdouble..distilledwateras ;eluent.TheCASpositivefractionswerecollected,and ;concentratedbylyophilization.Theconcentrated ;fractionwasredissolvedindouble-distilledwaterand ;furtherpurifiedwithaCMSepharoseFastFlow

    ;fPharmacia)column.Thecolumnwaswashedwith ;onebedvolumeofdoubledistilledwaterandtwobed

    ;volumesOfO.5molLNaCIsolution.

    ;2.4StructuralcharacteristicsofAn76

    ;siderophore

    ;TheArnowtest[201wasusedfordetectionofthe ;catecholatetypestructures,catecholateisyellowwhen ;reactedwithnitrousacid,andchangestoorangered

    ;whenNaOHsolutionisaddedtothemedium.2, ;3-Dihydyoxybenzoicacid(0.1mmol,Acros

    ;Organics)and0.1mmolcatecholwereusedas ;Siderophoreproductionfrom27f’damentonsfungalstrainsandanovelsiderophorewithp

    otentialbiocontrol

    ;applicationsfromAspergillusnigerAn76

    ;standardsforthisassay.Hydroxamatetypestructures ;weredeterminedbvthemodifiedCsakymethod[21.

    ;Anorange.redcolorindicatedtheexistenceof ;hydroxamates.Acetohydroxamicacid(O.1mmolL,

    ;ICNBiomedieals)and0.1mmo1L..hydroxylamine ;hydrochloridewereusedasstandards.Coomassieblue ;staining,ninhydrintest,andbiuretproteinassaywere ;usedtodetecttheaminoacidsandpeptidebondsin ;theAn76siderophore.Thepartiallypurified ;siderophorewashydrolyzedwith12molLHCIat ;110.Cfor24h,andthehydrolysatewasanalyzed

    ;withaHitachi83550highspeedaminoacidanalyzer. ;2.5Scavengingeffectsofhydroxylfree ;radicalsofAn76siderophoreinvitro

    ;Fentonreactionwasusedforproductionof ;H0.whichwasdetectedwiththethiobarbituricacid ;fTBA)method[22.1mLsiderophoresolutionwas

    ;addedto1mlFentonreagent,thenmixedwithlml ;citratebuffer(0.05mmolL,pH4.5)containing

    ;2Ddeoxyribose(Amresco,4mmo|L-),andFeel3 ;(0.1mmolL),incubatedat30.C,shakingfor2h, ;then1mlthiobarbituricacid(BioBasicInc,1%in50 ;mmolL’NaOHsolution)and1mltrichloroacetic

    ;acid(w/v,2.8%)wereadded.Themixturewasheated ;inboilingwaterfor15min.Andtheabsorbanceat ;532nmwasmeasured.Electronspinresonance(ESR) ;trappingstudywasalsousedfordetectionofthe ;scavengingactivitiesofhydroxylfreeradicalsof ;An76siderophore.5,5-dimethylpyrroline1oxide

    ;(DMPO,0.08molL,Sigma)wasusedasspin

    ;trapperforHO?inESRstudy.ESRspectrawere ;recordedatroomtemperatureusingBrukerESP300 ;ESRspectrometer.

    ;2.6AntibacterialactivitiesofAn76

    ;siderophore

    ;LBbrothwasinoculatedwithbacterialstrains ;andincubatedat37.C,withshakingat170rpmfor ;I2h.BacteriaIculturemediaweredilutedwith ;distilledwaterto2-3×1o4ce11sml.Thebacteria1

    ;suspensionwasmixedwith8mgmlsiderophoreat

    ;roomtemperaturefor30min,and100lofthe ;solutionwasspreadontoanLBplate.Sampleswere ;platedintriplicate.and0.9%NaC1solutionwasused ;asanegativecontro1.Plateswereincubatedat37.C ;overnight,andcoloniesontheplatescounted.The ;samemethodwasappliedusing4mgml,2mgml

    ;and1mgmlconcentrationsofsiderophore,

    ;incubatingatroomtemperaturefor90min.Liquid ;mediumdilutionmethodwasusedtodetectthe ;minimuminhibitoryconcentration(M1C)ofAn76 ;siderophore.

    ;3.Results

    ;3.1Screeningfungalstrainsforsiderophore ;production

    ;Weinvestigatedsiderophoreproductionamong

    ;27filamentousfungi,includingplantpathogenicfungi ;andlignocellulosedegradingfungi.Theywere

    ;culturedinthreedifferentmedia.AsshowninFig.1, ;allstrainscouldsynthesizeandexcretesiderophores, ;buttheircapacitiesofsiderophoreproductionwere ;differentondifferentmedia,anddependedontheir ;growthstatus.Onlyninestrainscouldgrowin ;minimalmedium,andtwoofthosestrains,Drechslera ;sp.andTrichodermapseudokoningii$38(fungal ;strainsNo.4and8),grewpoorlywithbarely ;detectablesiderophoreproduction.Theotherseven ;strains,AspergillusaculeatusSML22,A.nigerAn76,

    ;Curvulariasp.,Polyporussp.6,Rhizoctoniasolani, ;Stemphyliumsp.andVenturiainaequalis(fungal ;strainsNo.1,2,3,5,6,7and9)grewbetterin ;minimalmediumandshowedhigheryieldsof ;siderophores.Allthe27strainsgrewwellinthetwo ;completemedia,butshowedlowersiderophore ;production.Therelativecapacitiesofsiderophore

    ;productionwerehighestinminimalmedium,although ;itwasnotsuitableforgrowthofallfungi.Itiswell ;knownthatbiosynthesisofsiderophoresisregulatedby ;ironcontentinthemediumandtheirproductionis ;repressedunderironrichconditionsI2.Thesynthesis

    ;21

    ;Siderophoreproductionfrom27filamentousfungalstrainsand ;applicationsfromAspergillus

    ;anovelsiderophorewithpotentialbiocontrol ;nigerAn76

    ;andexcretionofsiderophoresisabiologicaladaptation ;phenomenonthatisobservedwhenmicroorganisms ;growunderiron-limitingconditions.

    ;Inour

    ;experimentsthetraceironIevelsinwheatbran ;mediumandpotatodextrosemediummayhave ;restrainedsiderophoresynthesis.Theseresults ;suggestedthatnutritionalcontentofthemediumhad ;impactonsiderophoreproductioninfungi. ;

    ;

    ;0

    ;=

    ;0

    ;

;0

    ;MimaImed~m

    ;SWheatbranmedium

    ;一埘m”8.

    ;BH

    ;jFig.1Comparisonofsiderophoreproduction ;inthreedifferentmedia

    ;Notes:FungalstrainsNo.:.Aspergillusaculeatus

    ;SML22;2,AspergillusnigerAn76;3,

    ;Curvulariasp.;4,

    ;Drechslerasp.;5,Polyporussp.6;6,Rhizoctoniasolani;7. ;Stemphyliumsp.;8,TrichodermapseudokoningiiS38;9, ;Venturiainaequalis;10,Colletotrichumphomoides;l1. ;CoHolussp.3;12,Exserohilumturcicum;13,Ganodermasp.13; ;14,Ganodermasp.14;15,Helminthosporiumsp.;16, ;PenicilliumdecumbensJuA10;I7,Penicilliumjanthinellum; ;18,Penicilliumsp.8;19,Phanerochaetechrysosporium14;20. ;Phanerochaetechrysosporium25;21,Pithomycessp.;22, ;Polyporussp.4;23,Polyporussp.7;24,Thelephorasp.10;25. ;Thelephorasp.12;26,TrichodennaaureovirideTVl:27.

    ;TrichodermareeseiQM9414.

    ;3.2Influenceofironconcentrationon

    ;siderophoreproduction

    ;Minimalmediumwasusedasthebasicmediumto

    ;evaluatetheeffectsofdifferentconcentrationsofferric ;iron(0,5,10,20,and40lumolL-Fe)onsiderophore

    ;22

    ;productionfromninefungalstrains(Fig.21.Drechslera ;sp.and7=pseudokoningii$38didnotsynthesize ;siderophoreswhenculturedon0and20pmolL-Fe?. ;Theresponsesoftheothersevenstrainscouldbe ;dividedintotwotypes;siderophoresynthesisofA.niger ;An76didnotchangewithdifferentironconcentrations, ;butsiderophoreproductionintheremainingsjxstrains ;reducedwithincreasingferriciron.

    ;concentration.In

    ;thosesixstrainssiderophoreproductionwasrepressedat ;20BmolLFe?.

    ;InStemphyliumsp.productionwas

    ;suppressedatironconcentrationsaslowas5pmol. ;Comparisonofsiderophoreproductionamongvarious ;strainsindifferentironconcentrationssuggestedthata ;lowconcentrationofironwasmoreconduciveto ;siderophoreproduction.Sincesiderophoreproductionin

    ;A.nigerAn76wascompletelyunaffectedbyiron ;concentration,thisfungalstrainwaschosentostudythe

    ;effectsofhigherironconcentrationsonsiderophore ;production.

    , ;,

    ;

    ;………

    ;:

    ;

    ;O’23?5’7

    ;I’.量,

    ;\-

    ;ot2345’7

    ;:::::::::::

    ;Fig.2EffectsofFe~concentration ;oilsiderophoreproduction

    ;Notes:0pmolL’(-),5IamolL’(?),10mol(v),

    ),40~tmolL(?). ;20pmolL(?

    ;I-0-I1l’0.I’|.IJIl’ll_l0

    ;Siderophoreproductionfrom27f’damentonsfungalstrainsandanovelsiderophorewithp

    otentialbiocontrol

    ;applicationsfromAspergillusnigerAn76 ;1.50

    ;1,2O

    ;0DI90

    ;V0,60

    ;3O

    ;O.oo

    ;O’2345

    ;Cultureme’da)}

    ;Fig.3EffectsofhigherconcentrationsofFe ;onsiderophoreproductioninAspergillusnigAn76

    ;Notes:0rnmo|L(?),0.1mmo]L(?),0.2mmolL

    ;(v),0.3mmolL(?),0.4mmolL(),0.6mmolL(?),

    ;0.8mmolL(),1.0mmolL.(?).

    ;一一一一

    ;O’2345

    ;CultureTimeday)

    ;Fig.4FormationofFewhenAspergillusnigerAn76was ;culturedwithhigherconcentrationsofFe3+ ;Notes:0mmolL-(-),0.1mmolL(?),0.2mmo|L

    ;(v),0.3mmolL-(?),0.4mmolL(),0.6mmolL(?),

    ;0.8mmolL’(),1.0mmolL-(?).

    ;Fig.3showedthatthesiderophoreproduction

    ;capacityofA.erAn76increasedwithdecreasing ;Fe”concentration,

    ;andmaximumproductionwas

    ;achievedat0mmolLFeH.

    ;Siderophoreproduction

    ;wasnotcompletelyinhibitedevenatiron

    .Kinetically, ;concentrationsashighas1mmolL

    ;siderophoresynthesisappearedtoexponentially ;increaseov6rthecultureperiod.Furthermore,itwas ;observedthatalargeamountofFe”formedinthe

    ;fungalculture(Fig.4).ThecapacitytoconveaFeto

    ;FeincreasedwithincreasingconcentrationofFe3in ;theculturemedium.

    ;3.3IsolationandpurificationofAn76 ;siderophore

    ;Theleadacetateprecipitationmethodwasused ;forpreliminaryisolationofAn76siderophore.The ;siderophorewaspartiallypurifiedbyaseriesof ;chromatographystepsusingSephadexLH20and ;CM?SepharoseFastFlowcolumns(Fig.5).Peak1 ;showninFig.5gaveflCASpositivereaction,and

    ;wascollectedforfurtherstudy.

    ;‘t

    ;

    ;0

    ;O306090120

    ;Elutionme(min)

    ;Fig.5CM-sepharosefastflowchromatogramof ;AspergillusnigerAn76siderophore

    ;3.4StructuralcharacteristicsofAn76 ;siderophore

    ;ThestructureofAn76siderophorewas

    ;determinedbytheArnowtestandthemodifiedCsaky ;method.Theresultsshownthatitwasneithera ;catecholatenorahydroxamate.Coomassieblue ;staining,ninhydrintest,andabiuretproteinassay ;shownthattherewasnoaminoacidorpeptidebond ;structureinAn76siderophore.Aminoacid ;compositionanalysisalsoindicatedthatAn76 ;siderophoredidnotcontainaminoacidsinits ;structure.

    ;3.5Scavengingeffectsofhydroxylfree ;radicalsofAn76siderophoreinvitro

    ;InTBAassay.2Ddeoxyriboseisoxidizedby

    ;HO?tomalonaldehyde.Theproductofmalonaldehyde ;andTBAhasacharacteristicabsorbanceat532nm ;whichisproportionaltotheamountofHO.Asshown ;inFig.6,An76siderophore(3and6mgml)could ;scaven?

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