Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis

ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Fusarium

culmorum

affects

expression

of

biofilm

formation

key

genes

in

Bacillus

subtilis

Maryam

Khezri

_,

_Gholamreza

_Salehi

_Jouzani

_,

_Masoud

_Ahmadzadeh

a_{MicrobialBiotechnologyandBiosafetyDepartment,AgricultureBiotechnologyResearchInstituteofIran(ABRII),AREEO,3135933151,}

Karaj,Iran

b_{DepartmentofPlantProtection,CollegeofAgricultureandNaturalResources,UniversityofTehran,Karaj31587-11167,Iran} c_{DepartmentofPlantProtection,FacultyofAgriculture,UrmiaUniversity,Urmia,Iran}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26November2014 Accepted28July2015

AssociateEditor:AndréRodrigues

Keywords: Bacillussubtilis Biofilm Sinr Tasa Fusariumculmorum

a

b

s

t

r

a

c

t

Itisknownthatthereiscorrelationbetweenbiofilmformationandantagonisticactivities ofBacillussubtilisstrains;but,themechanismofthiscorrelationisnotclear.So,theeffectof theplantpathogen(Fusariumculmorum)onthebiofilmformationinaB.subtilisstrainwith highantagonisticandbiofilmformationactivitieswasstudied.TheexpressionofsinRand

tasAgenesinvolvedinthebiofilmformationwasstudiedinbothsinglecultureofbacterium (B)andco-culturewithF.culmorum(FB)usingreal-timePCR.Theresultsrevealedthatthe expressionofthesinRgeneinbothBandFBconditionswascontinuouslydecreasedduring thebiofilmformationperiodand,after24h(B4andFB4),itreached1%and0.3%atthe planktonicphase(B1),respectively,whereastheexpressionofthetasAwascontinuously increasedandwas5.27and30timesmorethanthatattheplanktonicphase(B1)after 24h,respectively.So,theexpressionreductionrateforsinR(3times)andtheexpression increasingratefortasA(6times)weresignificantlyhigherinFBconditionsthantheBones. TherelativeexpressionofsinRinFB1(planktonicphase),FB2(8h),FB3(12h),andFB4(24h) timeswas0.65,0.44,0.35,and0.29,whereasthetasAgeneexpressionwas2.98,3.44,4.37,and 5.63-foldoftheoneatcoordinatetimepointsinBconditions,respectively.Thesignificant expressionreductionofsinRandincreaseoftasAconfirmedthatthepresenceofpathogen couldstimulatebiofilmformationintheantagonisticbacterium.

©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Bacillussubtilisisknownasoneofthemostimportant antago-nistic(biocontrolagent)andplant-growthpromotingbacteria (PGPR)thatisisolatedfromrhizosphereofdifferentkindsof

∗ _{Correspondingauthor.}

E-mail:gsalehi@abrii.ac.ir(G.S.Jouzani).

plants.1–4_{B.subtilisstrainshavethepotentialtoproducemore}

than twodozensofdifferentantimicrobialcompounds and antibioticswithanamazingvarietyofstructures5_andalsoare

abletoformmulticellularstructuresorbiofilm.6–8_Biofilm

for-mationoccursinmanybacterialspeciesinresponsetodiverse environmental conditions such as nutrient depletion and

http://dx.doi.org/10.1016/j.bjm.2015.11.019

1517-8382/©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

drought,anditismediatedbymanymechanical, biochemi-cal,andgeneticfactors.8–10_{Commonly,amixtureofpolymeric}

compounds(e.g.extracellularpolysaccharides,proteins,and DNA)andanaggregationofdifferentmicroorganismscanbe foundinbiofilms.11,12_{Abilitytoformbiofilmisassociatedwith}

numerousbenefitsforitsbacteria.Forinstance,antibioticsare themostcommontoolstoremovebacteria;but,theyarenot efficientinthebiofilmstructure.6,13,14

Biofilmformationdependsontwomatrix geneoperons, includingyqxM(tapA-sipW-tasAgenes)andepsA-O(15genes) which are directly controlled by a repressor SinR and are responsible forthe synthesisofamyloid-like fibers and an exopolysaccharide astwo major biofilm components.2,15–19

DerepressionistriggeredbysinIwhichisactivatedby phos-phorylated Spo0A (Spo0A∼P) as a master and important regulatory protein in biofilm formation process.2 _{The tasA}

geneintheoperonyqxMisthemajorgenewhichencodesthe proteininvolvedinantimicrobialactivities,sporecoat assem-bly,andgermination.Itisalsofoundinthestationaryphase, sporulatingcultures,andthebiofilmmatrix.16,17,20–22

The environmental conditions and presence of other organisms like plant pathogens, symbionts, commensal-ism organisms, and plant hosts can affect biofilm for-mation; therefore, different biofilm structures such as plaques,slimes, pellicles, andcoloniesare seenunder var-iousconditions.1,18,21,23,24 _{Previously,someresearchershave}

shownthatthereispositivecorrelationbetweenbiofilm for-mationaswellasPGPRandantagonisticactivitiesofB.subtilis

strains.1,2,25,26_Baisetal.1_{demonstratedthataB.subtilisstrain}

(ATCC6051)wasabletoformbiofilm-likestructuresonthe rootsofArabidopsisplantsandprotectArabidopsisfrom infec-tionsbyPseudomonassyringae.Chenetal.2_{showedthatplant}

protectionbyantagonisticB.subtilisstrainsagainstRalstonia solanacearumdependedonwidelyconservedgenesrequired forbiofilmformation,includingregulatorygenesandgenes formatrixproduction;so,theyprovidedevidencesuggesting thatmatrixproductioniscriticalforbacterialcolonizationon plantrootsurfaces.

Previously,we isolatedand selectedsomenative B. sub-tilisstrainswhichhadhigh biofilmformationpotentialand antagonisticcapabilityagainstFusariumculmorum,thecausal agent of foot and root rot on wheat. Finally, the strains withhighbiofilm productionand biocontrolpotentialwere selected.TheB.subtilisstrainBs12isolatedfromsugarbeet fieldsinKermanshahregion(Iran)showedhighbiofilm for-mation,volatileproduction,proteaseactivity,and79.4%and 83% inhibitoryeffect againstF. culmorum atlaboratory and greenhouselevels,respectively.27 _{Itwasshownthatvolatile}

andproteaseproductionaswellasbiofilmformationbythis strainandalsootherselectedstrainshadsignificantlypositive correlation with their antagonistic ability,27 _which

coordi-nated with the previous reports.1,2 _{The principal purpose}

ofthis investigation was to find apart ofthe mechanism forcorrelation betweenbiofilm formationand antagonistic effect at molecular level; therefore, the effects of a plant pathogenicfungus(F.culmorum)onformingbiofilminB. sub-tilis(Bs12)were evaluated.Todoso,expressionofthetasA

and sinR genes in the strain Bs12 was investigated using real-timePCRmethodbothinthepresenceand absenceof

F.culmorum.

Materials

and

methods

Microorganismsandcultureconditions

Thenative B. subtilis strainBs12(GenBank accession num-berHQ234328)withhighpotentialinbiofilmformationand antagonisticactivityagainstF.culmorumwasused.27_The

bac-terial strain was routinely grownon nutrient agar (NA) or Luria-Bertanibroth(LB)at37◦C.Forlongmaintenance,sterile 40%glycerolwasusedaccordingtoWellerandCooks28_and,

then,transferredto−20◦_{C.TheF.culmorumstrainwaskindly}

providedbyPlantProtectionResearchInstituteofIran(PPRI), cultivatedonpotatodextroseagar(PDA)at27◦Cforroutine experiments, and transferredto4◦C forlong time mainte-nance.

Primersdesigning

Two specificprimerspairs,TasA-F(CAAGCCGTTCCACTG TGTAG)/TasA-R(AACCGCTCCTGAATATGATGG)and SinR-F(AAAGGCTACTCACTATCAGAAC)/SinR-R(TCTAATTGA CCA TCGTATTCGG),were designedusingOligo (National BioscienceInc.,version5)softwareforconductingthe real-timePCRexperiments. Theseprimersamplified181bpand 188bpDNAfragmentsoftasAandsinRofB.subtilis, respec-tively.Theprimerpairs,16SrRNA-F(GTAACCTGCCTGTAA GACTGG)/16SrRNA-R(CTGTAAGTGGTAGCCGAAGC),with the PCRproductlengthof110bpwereusedastheinternal control.Primersweredesignedinordertohavethelengthof about20–22bases,G/Ccontentbetween40.9%and55%,and Tmofabout56–59◦C.LengthofthePCRsecondarystructures anddimerformationwascontrolledusingOligoAnalyzer1.0.3 software.TheprimersweresynthesizedbyMWG(Ebersberg, Germany).

Toevaluatethespecificityoftheprimers,aPCRwas per-formedusinggenomicDNAofB.subtilis(Bs12)andF.culmorum.

Genomic DNA ofthe bacterium and fungus was extracted usingGenEluteTM_{BacterialGenomicDNAKit(Sigma–Aldrich,}

Zwijndrecht,NL)andCore-oneTM_{kit(CoreBio,USA),}

respec-tively.

Co-cultureofB.subtilisandF.culmorum

Bs12cellsweregrowninbiofilmgrowthmedium(BGM) con-taininganLB-basedmediumplus0.15Mammoniumsulphate, 100mM potassiumphosphate, pH7,34mMsodiumcitrate, 1mMMgSO4,and0.1%glucose,asdescribedbyHamonand

Lazazzera.29_{Samplingwasperformedunderbacterial}

plank-tonicandbiofilmformationconditions,accordingtoStanley et al.11 _{To obtain planktoniccells, the bacterial cells were}

grown overnight in BGM mediumat37◦C withshaking at 200rpm. Afterwards, the medium containing bacteria was dividedintotwoparts,oneco-culturedwithsuspension con-taining106_{sporespermLofF.culmorumandanotherwithout}

anyfungaltreatment(asnegativecontrol).Bothbeakerswere put in the above growth conditions for three more hours (OD600=2.5forcontrol).Atthistime, thebacterialmedium

wasdilutedwithanOD600=0.1infreshmediumand,then,the

thesame amountoffreshmedium.Analiquot ofcontents ofeachbeakerasplanktoniccellpopulation(6mL)was har-vested by centrifugation at 8000rpm for 10min for RNA isolation. Inthe second step,both beakers were incubated at37◦Cwithoutshaking;theseconditionswerenecessaryto inducebiofilmformationinbacteria.Thenextsamples con-taining6mLtakenfromeachbeakerwereharvested8,12,and 24hafterincubationbycentrifugationat8000rpmfor10min. Allthetakensampleswereimmediatelyputat−80◦_Cuntil

RNAextraction.Tonormalizetheexperiments,100␮Lofthe mediacontainingmicroorganismswereculturedonNAand bacterialCFUwascountedineachsampleafter24hat37◦C. BeforeRNAisolation,normalizationwasperformedby dilut-ingthesamplescontainingmorebacterialcells asthefinal bacterialCFUwasthesameforallthetreatments.

TotalRNAisolationandcDNAsynthesis

TotalRNAwasextractedfrom theharvestedsamplesusing Aurum Total RNA Mini Kit (Bio-Rad, USA) according to the manufacturer’s instructions.The concentrationofRNA wasquantified using aspectrophotometer (NanoDrop 1000 spectrophotometer-Thermo Scientific). PCR was performed usingRNA(0.6␮g)asthetemplatetoensuretheabsenceof genomicDNAcontaminationintheRNAsamples.The tem-peratureprofileforPCRconsistedofafirstdenaturationstep of 5min at 94◦C, followed by 40 cycles of 94◦C/1min for denaturation,60◦C/1minforannealing,and72◦C/1minfor extension.Afinalextensionwascarriedoutat72◦C/5min.

Total RNA was transformed into cDNA using iScriptTM

cDNA SynthesisKit (Bio-Rad, USA)following the manufac-turer’sprotocol.ThetemperatureprogramforcDNAsynthesis was25◦C/5minfortheattachmentofprimers,42◦C/45minfor cDNAsynthesis,and85◦C/5minforenzymeinactivating.

Real-timePCR

Real-timePCRwascarriedoutusingiCycleriQrealtimePCR (Bio-Rad,USA)usingIQTM_SYBR®_{GreenSupermixKit(Bio-Rad,}

USA)in96-wellplates.AfterthedilutionofcDNA,1␮L(20ng) wasadded to24␮LofPCR mixture(12.5␮LofIQTM _SYBR®

GreenSupermix,1␮Lofeachprimerat10pmol/␮Land9.5␮L ofRNase-freewater).SpecificcDNAswereamplifiedby real-timePCRusingthespecificprimers.Thereal-timePCRcycling conditionsweredesignatedasfollows:initialdenaturationat 95◦Cfor2min,followedby40cyclesof95◦Cfor20s, 60◦C for30s,and72◦Cfor20s,andthefinalextensionwas car-riedoutat72◦Cfor5min.Fluorescencemeasurementswere recordedduringeachannealingstep.Toestablishamelting curveandconfirmtheprimers’specificity,anadditionalstep startingfrom50to95◦Cwasperformed.Thisstepincluded ninety10scycles, ineach ofwhichthere wastemperature increaseby0.5◦Cand, atthe end ofeach10s, the emitted fluorescencewasrecorded.Theefficienciesofamplifications weredeterminedbyrunningastandardcurve bytheserial dilutions ofcDNA. Efficiency can be calculated bythe for-mula:E=[10(1/−s)−1]×100,wheresistheslopeofstandard curve.Foreachmeasurement,athresholdcyclevalue(CT)was

determined.CT isdefinedasthenumberofcycles required

forthefluorescentsignaltopassthethreshold(i.e.exceeds

the backgroundlevel). Finally, theexpressionofgenes was calculatedbyformula2−Ct.30_{Theresultswerenormalized}

usingB.subtilis16SrRNAgeneastheinternalgene.The ultra-purewaterwasusedinsteadofcDNAasanegativecontroland thegeneexpressionlevelswerecomparedwiththenegative control.

Statisticalanalysis

MeasuresweretakenforeachconditionbycDNAsynthesized fromRNAextractedfromthreeindependentculturesand per-formedintriplicateforeachgene.Real-timePCRdataanalysis wasperformedusingBio-Radsoftwarebasedonthe thresh-oldcycle(CT).Analysisofvariance,comparisonofmeans,and

scoreoftreatmentgroupswereobtainedusingSAS(version 9.1)andDuncanMultipletest(p<0.01).

Results

Primerspecificityandreal-timePCRoptimization

Toevaluatespecificityofthedesignedprimers,PCRwas car-riedoutusinggenomicDNAofB.subtilis(Bs12)andF.culmorum.

WhenbacterialgenomicDNAwasusedasthetemplate, TasA-F/TasA-RandSinR-F/SinR-Rprimersamplified181and188bp fragments,respectively.Inaddition,thePCRproductof inter-nalcontrolprimerwasa110bpfragment.NoPCRproductwas observedwhenthefungalgenomicDNAornegativecontrol wasused.AftersamplingandRNAextraction,PCRwas per-formedusingthesamplesofRNAand16SrRNA-F/16SrRNA-R. Nofragmentwasamplifiedinthesamples.Theseresults con-firmed that there was no DNA contamination in the RNA samples. To determine the amplification efficiency, differ-entserialdilutionsofcDNAwereusedforeachprimer.For instance,fivedilutionsofcDNAfrom 1to0.0001wereused for 16SrRNA primersand, finally,cycle threshold, Tm, and standardcarverswereobtained.Accordingtothisexperiment, theefficiencyof16SrRNA,TasA,andSinRprimerswas deter-minedas92.75%,99.98%,and96.78%,respectively.

EvaluatingeffectofpathogenpresenceonsinRexpression

RelativeexpressionlevelsofsinRgeneinthestrainwere calcu-latedintheabsence(B)andpresenceofthefungus(FB)from threeindependentculturesintriplicate.Theresultsindicated thatthemaximumexpressionofsinRwasobservedwhenthe bacterialcellswereintheplanktonicphaseintheabsenceof

F.culmorum(B1)(Fig.1(a)).Byenteringthebiofilmformation phase, theexpressionofthe genewas criticallydecreased, whichwascontinuedoverthetimefromB1toB4(24hafter enteringthebiofilmformationperiod).Themaximum reduc-tionrateofthegeneexpressionwasobserved8hafterstarting thebiofilmformationcomparedwithplanktonicphase(about 80%reductions)andtheminimumexpressionwasobserved 24hafterstartingbiofilmformation(B4)whichwasabout1% oftheexpressionlevelinplanktonicphase(Fig.1(a)).When thebacterialcellswereco-culturedwithF.culmorum,thesinR

expressionreductiontrendwascriticallyincreased.Similar totheexperimentsinwhichplantpathogenwasabsent,the

1.2 1 0.8 0.6 0.4 0.2 0 1.2 a _a b b c c c d 1 0.8 0.6 0.4 0.2 0 Planktonic phase Relativ e e xpression Relativ e e xpression Planktonic phase

Times after starting biofilm formation phase Time after starting biofilm formation phase

8 h 12 h 24 h 8 h 12 h 24 h

a

b

Fig.1–RelativeexpressionofthesinRgeneduringbiofilmformationperiodcomparedwiththeplanktonicphase(a)inthe

singlecultureofB.subtilisstrain(B),(b)intheco-cultureofbacteriaB.subtilisandF.culmorum(FB).Differentlettersindicate

significantdifference(p<0.05).

maximumexpressionintheco-culturesystemoccurredwhen thebacterialcellswereinplanktonicphase(FB1).Itwas con-tinuouslyreduced8(FB2),12(FB3),and24(FB4)hafterentering thebiofilmformationphaseandreached3%oftheexpression levelinplanktonicphase(FB1)(Fig.1(b)).

Themaximumreductionrateofthegeneexpressionwas observed8hafterstartingthebiofilmformation(about64% reductions).Comparisonoftheresultsofbothtreatments(B andFB)showedthattherelativeexpressionofsinRinFB con-ditionwassignificantlylowerthanthatofBconditioninthe samegrowthconditionsandtimepoints(Fig.2(a)).The rela-tiveexpressionforFB1,FB2,FB3,andFB4was0.65,0.44,0.35, and0.29comparedwithB1,B2,B3,andB4,respectively,and byincreasingthetimeduringthebiofilmformationperiod, thereductionrateofthesinRgeneexpressioninFBcondition wascontinuouslyincreasedcomparedwiththatofB condi-tioninthesamegrowthconditionsandtimepoints(Fig.2(a)). TherelativeexpressionofthesinRgeneduringtheplanktonic andbiofilmformationperiodinBandFBconditionscompared

withtheplanktonicphaseofBconditionisshowninFig.3(a). Themaximumandminimumexpressionswereobservedin theplanktonicphaseofBcondition(100%)and24hafter start-ingthebiofilmformationinFBtreatment(0.3%),respectively (Fig.3(a)).

Evaluatingeffectofthepathogenpresenceonexpression oftasAgene

TheresultsofquantitativePCRshowedthattheexpressionof

tasAgenecontinuouslyincreasedfromplanktonictothefinal biofilm formationphasesinBcondition(B1–B4).The maxi-mumgeneexpressionwasobservedwhenthebacterialcells wereat24hafterstartingthebiofilmformationperiodinthe absenceofF.culmorum(B4),whichwas5.27timesmorethan thatintheplanktonicphase(B1)(Fig.4(a)).Byentering the biofilm formationphase,thegeneexpressionwascritically increased,whichwascontinuedoverthetimefromB1toB4 phases.Themaximumincreasingrateofthegeneexpression

7 6 5 4 3 2 1 0 0 Planktonic phase Relativ e e xpression Relativ e e xpression 8 h 12 h 24 h Planktonic phase

Time after starting biofilm formation phase Time after starting biofilm formation phase

8 h 12 h 24 h 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 a b c d d c b a

a

b

Fig.2–RelativeexpressionofthesinR(a)andtasA(b)genesduringplanktonicphaseandbiofilmformationperiodsinthe

co-cultureofB.subtilisandF.culmorum(FB)comparedwithitsexpressionpointwhenthepathogenisabsentateachtime

Planktonic phase Planktonic phase Relativ e e xpression Relativ e e xpression 8 h 12 h 24 h 8 h 12 h 24 h

Time after entering biofilm formation Time after starting biofilm formation phase

1.2 35 30 25 20 15 10 5 0 1 0.8 0.6 0.4 0.2 0 a a b b c c e d e d f g g gh g f B FB B FB

a

b

Fig.3–RelativeexpressionofthesinR(a)andtasA(b)genesduringbiofilmformationperiodinbothsingle(B)and

co-culture(FB)conditionscomparedwiththeplanktonicphaseinthesingleculturecondition.Differentlettersindicate

significantdifference(p<0.05). 0 Planktonic phase Relativ e e xpression Relativ e e xpression 8 h 12 h 24 h Planktonic phase

Time after starting biofilm formation phase Time after entering to biofilm formation phase

8 h 12 h 24 h 1 2 3 4 5 6 3.5 3 2.5 2 1.5 1 0.5 0 4 a b c d d c b a

a

b

Fig.4–RelativeexpressionofthetasAgeneduringbiofilmformationperiodcomparedwiththeplanktonicphase(a)inthe

singlecultureofB.subtilisstrain(B),and(b)intheco-cultureofbacteriumB.subtilisandF.culmorum(FB).Differentletters

indicatesignificantdifference(p<0.05).

wasobservedat24hafterstartingthebiofilmformationin whichtheexpressionofthegenewasincreasedupto315% comparedwiththephaseB3,whereastheminimum increas-ingratewasobservedat8hafterstartingbiofilmformation (B2)inwhichthe expressionofthe genewas increasedby about18%comparedwiththepreviousphase(B1)(Fig.4(a)).

Whenthe bacterialcellswere co-cultured withF. culmo-rum,thetasAexpressionwassignificantlyandcontinuously increased, which was critically increased during different phasesfromFB1toFB4(Fig.4(b)).Thisincreasingratewas sig-nificantlymorethanthatofthetreatmentscontainingonly bacterialcells.ThelevelsofexpressionofthetasAgene8,12, and24hafterenteringbiofilmformationphasewere1.59,2.99, and3.26timesmorethanthoseoftheplanktonicphase(FB1) (Fig.4(b)).

TheexpressionofthetasAgeneinFB1toFB4phaseswas 2.98,3.44,4.37,and5.63-foldoftheoneatsimilartimepoints inB(B1toB4)conditions,respectively(Fig.2(b)).Theseresults suggestedthatthepathogenicfungusstimulatedthe expres-sionoftasAgene.Fig.3(b)showstherelativeexpressionofthe

tasAgeneduringtheplanktonicandbiofilmformationperiods

intreatmentsBandFBcomparedwiththeplanktonicphaseof treatmentB(B1).Themaximumexpressionwasobserved24h afterstartingthebiofilmformationintreatmentFB,whichwas 30timesmorethanthatoftheplanktonicphaseoftreatment B(3000%increase)(Fig.3(b)).

Discussion

Wecharacterized30IraniannativeB.subtilisstrainsisolated from the rhizosphere of various hosts in different regions of Iran. The results of laboratory and greenhouse experi-mentsshowedthatvolatileandproteaseproductionaswell asbiofilmformationbysomestrainshadsignificantlypositive correlationwiththeirantagonisticabilityand,finally,themost powerfulantagoniststrainswithhighbiofilmproductionwere selected.StrainBs12isolatedfromsugarbeetfieldsin Kerman-shahregionshowed79.4%and83%inhibitoryeffectagainst

F.culmorumatlaboratoryandgreenhouselevels,respectively, andhighbiofilmformation,volatileproduction,andprotease activity; therefore, it was selected forthe present study.27

Previously, it has been shown that many different factors suchas different fungal compounds (fungal culture super-natant), pH, temperature, nutrient compounds,31 _indole,13

complexpolysaccharides,32_{andoxygenrateaffectthebiofilm}

formation.So,toexplorethedetailedmechanismsof differ-entfactorsonbiofilmformationinB.subtilis,itisnecessary to perform detailed studies covering all bioticand abiotic environmentalfactors.Different geneticpathwaysthat are inducedbyenvironmentalsignalsareinvolvedinthe inter-action of cells and abiotic surfaces. These factors can be changedintheamountortypeofnutrientcontent,osmotic factor,pH,temperature,iron,oxidativestress,andsubstrate type.33,34_{StanleyandLazazzera}35_showedthat

environmen-talsignalsand regulatoryproteinsaffecttheinitialstepsof bacterialbiofilmformationandthenatureofmaturebiofilm structure.So,surfaceattachment andbiofilmformationon differentbioticandabioticsubstratesareinfluencedbynature andvarious environmentalstimulations.24 _{Thepresenceof}

otherorganisms,suchaspathogens,isknownasoneofthe factors affecting the biofilm formation and structurein B. subtilis;but,themechanismisnotwellknown.1,25,26_So,the

principalpurposeofthisinvestigationwastofindapartof themechanismforcorrelationbetweenbiofilmformationand antagonisticeffectatmolecularlevel;therefore,theeffectsof aplantpathogenicfungus(F.culmorum)onformingbiofilmin

B.subtilis(Bs12) were evaluated.Todoso,real-time PCRas asensitiveandquantitativetechniquewasusedtomeasure theexpressionprofilesoftwoimportantgenes(sinRandtasA)

involvedinthebiofilmformationprocessofthebacteriain thepresenceandabsenceofF.culmorum,thecausalagentof wheatcommonrootrot.B.subtilisiscommonlyisolatedfrom rhizosphereofdifferentplants,showsantagonisticactivities againstplantpathogens,and maybeusedasplant-growth promotingbacteria.1,3,4_{Variousmicroorganismscanbefound}

togetherintherhizosphere.Thepresenceandproductionof metabolitesbyothermicroorganismscanbeveryeffectivefor biofilmformationintargetbacteria.Ourresultsindicatedthat theexpressionofsinRwassignificantlyreducedinthe pres-enceofthepathogenicfungus.Expressionofthisgenewas atahigh levelintheplanktonicphaseofbacterialgrowth; but,itdecreaseduponenteringtheproductionofbiofilm,as was expected. Several previousstudies have demonstrated thatsinRasoneofthemostimportantregulatorygeneshasa directnegativecontrolonbiofilmformationofB.subtilis.17,20,21

Leimanetal.36_{showedthatpointmutationsinthesinRgene}

resultedina significant increaseinbiofilm formation inB. subtilisandconfirmedthatitisakeymatrixregulatorygene forbiofilmformation. Previously,thissubjecthasbeenalso confirmedbyotherresearchers.37,38_{Synthesisofmain}

compo-nentsofbiofilmmatrix,suchasextracellularpolysaccharides andproteins,ismediatedbytwooperonsof15-geneepsand three-gene yqxM,respectively.16 _{Both of these operons are}

underdirectnegativecontrolofthesinR gene.Indeed,this repressorproteinbindstomultiplesiteswithinthepromoter regionforthementionedoperons,therebyrepressingits tran-scription.Whenthisnegativeregulatorisactive,expression ofthese18importantgeneswillbesuppressed.So,sinRgene isknownasamasternegativeregulatorinthebiofilm forma-tionprocessofB.subtilis.17_{TranscriptionofthesinRgeneis}

controlledbyanothergenecalledsinI.Whenbacteriaarein

biofilmformationconditions,suchasenvironmentalstress, shortageofsomenutrient sources,etc.,transcriptional fac-torSpoAbecomesphosphorylatedandactivatesexpression ofsinI.ActivatedsinIcanbepreventedfromsinRexpression. Thus,epsandyqxMoperonsareactivatedand,consequently, the genesinvolvedinbiofilm formationareexpressed.20 _In

the present study, it was observed that the expressionof

sinRwasdecreasedinthebiofilmcomparedwiththephase ofplanktonic cells inbothtreatments. On theother hand, expressionofsinRinfree-swimmingcellswashigherthanthat ofthesessileonesinthepresenceorabsenceofF.culmorum.

TheexpressionofsinRinFBconditionwaslessateachtime pointcomparedwithBcondition.Inplanktoniccells,thegene expressionlevelwasdecreasedintheco-culturesystem(FB1) comparedwithsinglecultureofbacterium(B1).This diminu-tionratewasrepeatedateachtimepointofbiofilmformation phase,namelyFB2,FB3,andFB4samplescomparedwithB2, B3,andB4samples,respectively.Theseresultssuggestedthat thepresenceoffungusinBs12growthmediumcauseda sig-nificantreductionofsinRgeneexpressioninbothplanktonic andsessilecells.

InthecaseoftasAgene,opposite resultswereobtained. Inthe planktonicphase,expressionofthegenewasatthe lowest level. By entering thebiofilm productionphase, the expressionofthetasAgenewasincreasedand,atfinaltime pointofbiofilmformation(B4),itreachedthehighestlevels of5.28-fold.Previously,someotherresearchershavereported theseresultsandshownsignificantincreaseintheexpression oftasAgeneduringbiofilmformation.20,39,40_{Thisincreasing}

ratewasobservedinbacterialgrowthphasesinbothsingle andco-cultureconditions;but,theincreasingrateinFBwas significantlymorethanthatoftreatmentsB.Brandaetal.16

showedthatTasAisamajorproteininbiofilmextracellular matrix and the absence of this proteinresults in a resid-ualmatrix.TasAhasbeendetectedinstationaryphaseand sporulatingcultures.ItappearsthatTasAhasseveralother functions;forinstance,itactsasabroad-spectrum antibac-terialfactorandseemstohaverolesinsporecoatassembly and germination.40,41 Evaluation of the relative expression of tasAgene showed that the expressioninFB treatments was significantlyincreasedcomparedwithBtreatmentsat eachtimepoint.Similarresultswereobservedinplanktonic andallbiofilmformationlevels.Basedonthesedata, expres-sion oftasA gene increasedin the presenceof pathogenic fungus. Many transcriptionalfactors inphysiological activ-ities ofbacteria are regulated byenvironmental stress;for instance,biofilm formationbyB.subtilisstimulatedin non-optimal growth conditions. As F. culmorum is a pathogenic fungus, its presence in growth medium of the antagonist bacteriahasprovidedanon-optimalcondition;consequently, inducedandenhancedbiofilmformationdown-regulatesinR

andup-regulatingtasAgenes.

Inadditiontothementionedkeygenesinvolvedinbiofilm formationinB.subtilis,ithasbeenrecentlydemonstratedthat otherdifferentgenesandfactorsaffectbiofilmformation.For instance,ithasbeenshownthatthegenesencoding antimi-crobialproteins,suchassurfactinandbacillomycinwhichare involved inthe antagonisticactivities ofbacteriumagainst plant pathogens, significantly and positively affect biofilm formation.25,26 _{Gerwig et al.}42 _{confirmed that the protein}

tyrosinekinasesEpsBandPtkAdifferentiallyaffectedbiofilm formationinB.subtilis.

Also, it has been shown that the RapP-PhrP Quorum-sensingsystemofB.subtilisaffectsbiofilmformationthrough multipletargets dueto anatypical signal-insensitiveallele of RapP.43 _{Recently, complete genome of some}

biofilm-formingB.subtilisstrainswithantagonisticactivitieshasbeen sequenced. Also,moredetailed information about the cor-relation of antagonistic activities and biofilm formation is expectedtobeexplored.44

Inconclusion,accordingtoourresults,forthefirsttime, itwasshownthatthemajorgenes,includingsinRandtasA

involvedinbiofilmformationinB.subtilis,weresignificantly affectedbytheinteractionofbacteriaandfungus.Also,the presenceof F. culmorum stimulated biofilm formation inB. subtilis.Thesefindingsconfirmedthatthepresenceofother organisms,suchasplantpathogensintheenvironmentofthe bacterium,stimulatedbiofilmformation.Thepresentstudy couldbethefirststeptodeterminethemechanismof relation-shipbetweenantagonisticactivitiesandabiofilmformation. But,tocharacterizethedetailedmechanisms,itisnecessary toperformmoredetailedstudiesinthisfield.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors wish tothank all the staff of Department of MicrobialBiotechnologyandBiosafety,Agricultural Biotech-nologyResearchInstituteofIran(ABRII),fortheir technical assistance.Thisworkwassupportedbyagrantfrom Agricul-turalResearch,EducationandExtensionOrganization(AREEO) (Grantnumber:2-05-05-8711).

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