Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation

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Bacterial

Molecular

Pathogenesis

Roles

of

quorum

sensing

molecules

from

Rhizobium

etli

RT1

in

bacterial

motility

and

biofilm

formation

Swarnita

Dixit

_,

_Ramesh

_Chand

_Dubey

_,

_Dinesh

_Kumar

_Maheshwari

_,

Prahlad

Kishore

Seth

_,

_Vivek

_K.

_Bajpai

a_{DepartmentofBotanyandMicrobiology,GurukulKangriUniversity,Haridwar,Uttrakhand,India} b_{BiotechPark,SectorG,Jankipuram,KursiRoad,Lucknow,UP,India}

c_{DepartmentofAppliedMicrobiologyandBiotechnology,SchoolofBiotechnology,YeungnamUniversity,Gyeongsan,Gyeongbuk,}

RepublicofKorea

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Articlehistory:

Received10June2015 Accepted31August2016 Availableonline8July2017 AssociateEditor:CristianoGallina Moreira Keywords: Rhizobiumetli Quorumsensing Motility Biofilm Lensculinaris

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StrainRT1wasisolatedfromrootnodulesofLensculinaris(alentil)andcharacterizedas

Rhizobiumetli(aGram-negativesoil-bornebacterium)by16SrDNAsequencingand phyloge-neticanalysis.ThesignalingmoleculesproducedbyR.etli(RT1)weredetectedandidentified byhigh-performanceliquidchromatographycoupledwithmassspectrometry.Themost abundantandbiologicallyactiveN-acylhomoserinelactonemolecules(3-oxo-C8-HSLand

3-OH-C14-HSL)weredetectedintheethylacetateextractofRT1.Thebiologicalroleof

3-oxo-C8-HSLwasevaluatedinRT1.Bacterialmotilityandbiofilmformationwereaffectedor

modifiedonincreasingconcentrationsof3-oxo-C8-HSL.Resultsconfirmedtheexistenceof

cellcommunicationinRT1mediatedby3-oxo-C8-HSL,andpositivecorrelationswerefound

amongquorumsensing,motilityandbiofilmformationinRT1.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Severalendophyticbacteriaresidewithinthelivingtissuesof hostplantsandcarryoutanumberofbeneficialfunctions.1

RhizobiumetliisaGram-negative␣-proteobacteriumfoundin soilthatformsrootnodulesinlentils(LensculinarisMedik.) andinotheragriculturallyimportantlegumes,suchas Phase-olusvulgaris.L.culinarisisaneconomicallyimportantlegume cropcultivatedintropical,sub-tropical,andtemperateareas worldwide.2 _{Many endophytic bacterial isolates(RT1–RT27)}

havepreviouslybeenscreenedandcharacterizedfromlentil rootnodules.3

∗ _{Correspondingauthor.}

E-mail:vbajpai04@yahoo.com(V.K.Bajpai).

Swarmingmotilityenablesgroupsofbacteriatomovein a coordinated manner on solid surfaces, and the swarm-ing phenomenon has been demonstrated for a number of bacteria,includingthemembersofAzospiriillum,Aeromonas, Burkholderia,Bacillus,Chromobacterium,Clostridium,Escherichia, Proteus,Pseudomonas,Rhizobium,Salmonella,Serratia, Sinorhizo-bium,Vibrio,andYersinia.4–7_{Bacterialmotilityprobablyoccurs}

asa dispersalstrategy innutrient-limitedenvironments or forlocatinganewhost.8,9 _{Normallyswarmingrequires the}

differentiationofvegetativecellsintoaspecializedcelltype called swarmercells.Surface contact(changein viscosity), physiological parameters, and chemicalsignals (nutritional status)providethestimulithattriggerthedifferentiationof

http://dx.doi.org/10.1016/j.bjm.2016.08.005

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

swarmercells.Although,pathwaysresponsibleforsignal inte-grationarepoorlyunderstood,itisbelievedthatsignalsare sensedand transmittedbytwo-component regulatory sys-tems, cytosolic regulators or flagella leading to a complex network.Differentiatedswarmercellsareoftenhyper flagel-latedandelongated,andthesecellsmoveingroupsorrafts organizedin parallelto the long axisof cells tomaximize cell–cellcontactand maximallycolonizeavailablesurfaces. Themigrationfrontisprecededbyavisiblelayerofslime-like extracellularmaterial,andasaresultofembeddinginthis extracellularslimematrix,populationdensitiesarenormally extremelyhigh.10

Bacteriamaydevelopwithinplantrootsasisolatedcells, micro-colonies,aggregates,orbiofilms.11_Biofilmsare

bacte-rial communitiessurrounded byaself-produced polymeric matrixattachedtoinertorbioticsurfaces.12_{Bacterialsurface}

components, particularlyexopolysaccharides (EPS),flagella, andlipopolysaccharides(LPSs),incombinationwithbacterial functionalsignals,arecrucialfortheformationofrhizobial biofilmsinallspeciesstudiedtodate,13andpolysaccharides atrhizobialsurfacesplayimportantrolesinsymbiosisandin formationofactiverootnodules.

Bacteriacanmonitorandrespondtochangesin environ-mentalconditionsviaacell density-related processknown as quorum sensing.14 _{Quorum sensing (QS) is a form of}

cell–cell communication regulated at the gene expression level and employed by several bacterial species to coordi-nate behavior at a community level. QS depends on the production of various signaling molecules, though N-acyl homoserinelactones(AHLs)aremostcommonlyutilizedby Gram-negativebacteria.Furthermore,thepresenceof AHL-likesignalmoleculesinGram-negativebacteriamaybedueto theactiveparticipationoftheLuxlfamilyofAHLsynthases, whichdifferinsidechainlength,degreeofsubstitution,and saturation.15_{ThephenotypesregulatedbyAHLsinRhizobium}

species are remarkably diverse and of profound biological andecologicalsignificance.Thesephenotypesinclude motil-ityandbiofilmformation,theproductionsofantibioticsand exo-enzymes,synthesisoftheplantgrowthpromotingauxin indole-3-aceticacid (IAA),thepromotion ofplant coloniza-tion, and biocontrol against several plant diseases. Some previousreportshavedemonstratedtheproductionofQS sig-nalingmoleculesbyvariousrhizobia,14,16_{butlittleisknown}

regardingthemechanismsofcommunicationamong lentil-nodulatingstrains.

Hence,theobjectiveofthepresentstudywastoidentify andcharacterizeQSsignalingmoleculesproducedbyR.etli

RT1andtoevaluatethebiologicaleffectsofoneAHLmolecule, namely,3-oxo-C8-HSLonprocessesrelatedtocell

communi-cation.

Materials

and

methods

Microorganisms

TheisolatesRT1–RT27werescreenedfromtherootnodules ofL.culinariscollectedfromafarmer’sfieldinUttarPradesh (India).IsolateRT1wasidentifiedasRhizobiumspeciesbased

onitsphenotypicandbiochemicalcharacteristics.3_Since

iso-lateRT1hadthebestplantgrowthpromoting(PGP)attributes, itwasfurtherstudiedindetail.RT1wasgrownat30◦Cfor24h intryptonesoyandyeastextractmannitolmediaforfurther use.

GenomicDNAextraction

GenomicDNAofRT1wasextractedasdescribedbySambrook and Russel (2001).17 _{Briefly, a bacterial culture was grown}

overnight in5mLtryptonesoybroth,stirredat150rpmfor 24hat30◦C.Theculturewasthencentrifuged at10,000×g

for 2min and the pellets so obtained were re-suspended in 50␮L 1× TE buffer (10mM Tris–HCl, 1mM EDTA), cen-trifugedat10,000×gfor2min,re-suspendedin350␮L1×TE buffer containing5␮Llysozyme(50mg/mL)and2␮LRNAse A(10mg/mL),andincubatedat37◦Cfor10min.Finally,3␮L proteinase K (20mg/mL) and 30␮L SDS (10%) were added and incubatedat37◦Cfor35min.ThecrudeDNAtemplate wasPCI(phenol–chloroform–isoamylalcohol)(25:24:1)andCI (chloroform–isoamylalcohol)(24:1)extracted,precipitatedin 1mLabsoluteethanol,washedwith500␮L70%ethanol, re-suspendedin50␮Lofsterilizedultrapurewater,andstoredat −20◦_C.

16SrRNAgeneamplification

Theamplificationofa1492bpregionofthe16SrRNAgenewas performedusing5-GGCTCAGAACGAACGCTGGCGGC-3asthe forwardprimerand5-CCCACTGCTGCCTCCCGTAGGAGT-3as thereverseprimer.18_{ThePCRprocedureconsistedof}

prepar-ingamastermixcontainingpolymerasebuffer(2.5␮L),10mM MgCl2(1.5␮L),10mMdNTP(1.2␮L)andsteriledistilledwater.

Thegenefragmentwasamplifiedusing5–25ngofbacterial DNA,100ngofeachprimer mentionedaboveand 0.3␮Lof

Taq polymerase(1U/␮L).Thereactionvolumewasadjusted to25␮Lusingsteriledistilledwater.Thechemicalsusedfor PCRwere acquired from BangaloreGenei(India). The reac-tion conditionsusedwere:initialdenaturationfor7minat 94◦C,then29amplificationcycles{denaturationfor1minat 94◦C,extensionfor1minat72◦C,annealingat54◦Cforseven cycles,at53◦Cand52◦Cforonecycleeach,andat51◦Cfor20 cycles}andafinalextensionfor10minat72◦C.Theamplified genewasvisualizedin0.8%agaroseafterelectrophoresis,and thePCRproductwassequencedattheInstituteofMicrobial Technology(IMTECH),Chandigarh,India.

Phylogeneticanalysis

Phylogenyandevolutionaryanalysiswere carriedoutusing theMEGA6.0package.19_{The16SrDNAsequenceofRT1was}

alignedusingCLUSTALW20_{againstcorrespondingnucleotide}

sequencesofRhizobium(typestrain)retrievedfromGenBank (NCBI). Theevolutionarydistancematrix was generatedas described by Jukes and Cantor (1969),21 _{and the}

phyloge-netictreewasinferredbytheneighbor-joiningmethod.22_Tree

topologieswereevaluatedbybootstrapanalysisbasedon1000 re-samplingsoftheneighbor-joiningdataset.

Nucleotideaccessionnumber

Thesequence of16S RNAgene ofRT1hasbeen deposited intheGenBankdatabaseunderaccessionnumberR.etliRT1 (KC762618).

ExtractionanddetectionofQSsignalmolecules

AHLmolecules were extractedas described byShaw etal. (1997).23 _{R. etli RT1 was grown in 500mL of AB minimal}

mannitol liquid medium for 48h to the stationary phase. Aftercentrifugationat7400×g,thesupernatantwasextracted twicewithanequalvolumeofethylacetatecontaining1.5mL ofacetic acid perliter.Theextractwasthenevaporated to drynessusingavacuumrotatorat42◦Candre-dissolvedin asmall volumeof ethylacetate. Thisculture extractof R. etliRT1wasthenanalyzedbyliquidchromatography/positive electrosprayionization(ESI+_{)multipleMS.Liquid}

chromatog-raphyandelectrospraymassspectrometry(LC-ESI–MS)were conductedusingaThermoFinniganLCQadvantagemaxion trapmassspectrometerandaFinniganSurveyorHPLCsystem. Thecolumnwasthermo ODS-2, 250×4.6, 5␮m andeluted withacetonitrile and waterat 800␮L/min. A 20␮L sample was introduced into the ESI source using a Finnigan Sur-veyorAutosampler.Massspectrawerescannedintherange of150–2000Th.Maximumioninjectiontimewassetat150ns, ionsprayvoltageat5.3kV,andcapillaryvoltage at40VA.A generalelutionprogramwasusedtoeluteallputativeAHLs.

Bacterialmotilityassay

Forthe swarmingexperiment,yeastextractmannitol (con-taining0.4g/LMgSO4·7H2O)softagarplates(0.75%;60/15mm)

supplementedwith3-oxo-C8-HSLatdifferentconcentrations

(0.05, 0.5, or 5.0␮M), were driedat 16◦C for5h and spot-inoculatedcentrally on theirsurfaces withthe appropriate RT1culture(1.2␮L,ODofovernightculture5950.7).Swarming behaviorwasanalyzedonswarmerplatescontaining 3-oxo-C8-HSL.Inoculatedplateswereincubatedat30◦Cfor24h,24

andswarminghalozonesweremeasured.Experimentswere performed4times.

Biofilmformationassay

Biofilmformationwas assayed quantitativelyas previously describedbyO’TooleandKolter(1992)25_{bystainingbiofilms}

with crystal violet. RT1 was grown in 96-well polystyrene platesat30◦Cfor24hwithoutshakingintrypticsoybroth (TSB)mediumcontaining0.05,0.5,or5.0␮Mof3-oxo-C8-HSL.

Planktoniccellswerethengentlyremoved,and180␮Lofan aqueoussolutionofcrystalviolet(0.1%,w/v)wasaddedfor 15min.Eachcrystalviolet-stainedwellwasthenrinsedthree timeswithsteriledistilledwaterandscoredforbiofilm for-mation by adding 150␮L of95% ethanol. Opticaldensities ofsolubilizedcrystalvioletwere measuredat560nmusing a Micro ELISA Auto Reader (Biotek).Non-inoculated YEMB mediumwasusedasacontrol.Controltubeswerevortexedfor 30s,andtheinitialopticaldensitywasdeterminedat600nm. Experimentswereperformed4times.

Effectof3-oxo-C8-HSLonthegrowthofRT1

Abacterialbrothco-inoculationassaywasperformedto visu-alizetheeffectof5␮M3-oxo-C8-HSLintrypticsoybroth(TSB)

mediuminordertoconfirmitseffectonthegrowthrateofRT1. Controlsetswerepreparedwithoutinoculating3-oxo-C8-HSL

andopticaldensitiesweremeasuredat600nm.

Statisticalanalysis

One-way analysisofvariance(ANOVA)followed bypost hoc

Fisher’s least-significant difference (LSD) test was used to determine the significances of the different effects treat-ments.All statisticalanalyses wereperformed usingPrism version6.03.

Results

and

discussion

Phylogeneticanalysis

Sequencing of the 16S rRNA gene provided a1492bplong sequence ofRT1. Aphylogenetic treeconstructedbasedon this sequence showedclustering ofthe RT1isolatewithR. etli CFN 42 (confidence=99%) (Fig. 1), and thus, RT1 was tentativelyclassifiedasR.etli.Basedonmorphologicaland bio-chemicalcharacteristics,3_{andcomparativeanalysisofthe16S}

rRNAgenesequence,thestrainwasunequivocallyidentified asR.etliRT1.Itisgenerallyacceptedthatorganismsdisplaying 16SrRNAgenesequencesimilarityvaluesof≥97%belongto thesamespecies.26_{Therefore,RhizobiumRT1wasconsidered}

tobeR.etli,sinceitexhibited99.3%similaritywithR.etliCFN 42.

Liquidchromatographyelectrospraymassspectrometry

(LC–MS)

The AHL molecules 3-oxo-C8-HSL and 3-OH-C14-HSL were

detectedintheextractofRT1.Therelativeabundance(RA)of 3-oxo-C8-HSLwas100%ataretentiontimeof9.82–10.07min

(Fig. 2), and that of 3-OH-C14-HSL was 20% at an RT of

14.36–14.62min.Comparativeanalysis ofthe AHLprofileof RT1byusingLC–MSshowedthepresenceofabroadrange ofAHLmoleculeswithdifferentacylchains.AHLmolecules are widelyknowntobeintercellularcommunicationsignal moleculesinbacterialpopulations.27_{Severalresearchershave}

reportedthepresenceofAHL-likesignalmoleculesinan Acine-tobacterstrainsthatthestrainsareabletoproducebiofilms andthatAHL-likemoleculesplayasubstantialroleinquorum sensinginthecontextofmetaltolerance.28,29

Motilityassay

ThemotilityofRT1increasedonincreasingtheconcentration of3-oxo-C8-HSLmolecule.Ataconcentrationofonly0.05␮M,

3-oxo-C8-HSLsignificantlyincreasedmotility,andathigher

auto-inducerconcentrations(0.5or5.0␮M)motilityincreased dose-dependently(Fig.3).Microorganismswiththeabilityto move are moreefficient atfinding morefavorable or less-hazardous niches forcolonization intheir environments,27

Rhizobium tropici CIAT 899(T) Rhizobium freirei PRF 81(T)

Rhizobium miluonense CCBAU 41251(T) Rhizobium leucaenae USDA 9039(T) Rhizobium paranaense PRF 35(T) Rhizobium vallis CCBAU 65647(T)

Ensifer numidicus ORS 1407(T) Ensifer sesbaniae CCBAU 65729(T) Rhizobium endophyticum CCGE2052(T)

Rhizobium grahamii CCGE 502(T)

Rhizobium tubonense CCBAU 85046(T) Rhizobium etli CFN 42(T)

Rhizobium sp. RT1

Rhizobium leguminosarum USDA 2370(T) Rhizobium llaguerreae FB206(T)

Rhizobium phaseoli ATCC 14482(T) Rhizobium pisi DSM 30132(T)

Rhizobium mesosinicum CCBAU 25010(T) Rhizobium sullae IS 123(T)

Rhizobium mongolense USDA 1844(T)

Rhizobium rhizoryzae J3-AN59(T) Rhizobium flavum YW14(T)

Rhizobium halotolerans AB21(T) 0.005

Fig.1–Neighbor-joiningphylogenetictreebasedon16SrDNAgenesequences,showingtherelationshipbetweenstrain RT1andthemostcloselyrelatedtypestrainsofRhizobiumetliCFN42.Thebarrepresents0.005substitutionspernucleotide position.

andalthoughmotilityinrhizobiaisnotessential,itisneeded to enable bacteria to find a specific host legume.27

Evi-dencehasbeenpreviouslypresentedregardingaconnection betweenquorumsensingandmotilitycontrolinR.etli,6_andin

Pseudomonasfluorescens.30_{Inaddition,Ahmer(2004)reported}

thataquorumsensingsystem(theLuxS/AI-2system)present inEscherichiacoli,31_{synthesizedAI-2toincreasemotilityand}

stimulatebiofilmformationbywild-typeE.coliK-12strain.32

Biofilmformationassay

On polystyrene RT1 developed a sessilebiomass with val-ues rangingfrom0.4to∼2.0ODat570nminthepresence ofexogenous 3-oxo-C8-HSL (Fig. 4),and athigh

concentra-tion (5␮M), 3-OH-C12-HSL caused a significant 3- to 5-fold

increase inbiofilm formation. Lowerconcentrations (0.5 or 0.05␮M)hadslighteffectsonbiofilmformation(Fig.4).When

3-oxo-C8-HSL

3-OH-C14-HSL

100

100

80

80

60

60

40

40

20

20

0

0

200

200

400

m/z ratio

m/z ratio

Relative abundance

288

298

Relative abundance

215

272

302

₃₂₉

485

262

245

199

227

171

300

Fig.2–LC–MSspectrashowingtherelativeabundancesofthemolecularionsofthemostabundantAHLmolecules, 3-oxo-C8-HSL(a)and3-OH-C14-HSL(b)inR.etliRT1supernatantafter48hofgrowth.

6 5 4 3 2 1 0 Control 0.05 Halo diameter (cm) 3-oxo-C8-HSL (µm) 0.5 5

Fig.3–Effectof3-oxo-C8-HSLonswimmingmotilitybyR.

etliRT1.Valuesaremeansofexperimentsperformedin quadruplicate.BarsrepresentSEMs(p<0.05).

1.5 1 0.5 0 Control 0.05 3-oxo-C8-HSL (µM) OD 570 nm 0.5 5.0

Fig.4–Effectofdifferentconcentrationsof3-oxo-C8-HSL onbiofilmformationbyR.etliRT1after24hofgrowth. Valuesaremeansofexperimentsperformedin quadruplicate.BarsrepresentSEMs(p<0.05).

exogenous3-oxo-C8-HSLwasadded0.05␮Manon-significant

3-foldincreaseinbiofilmformationwasobservedversusthe non-treatedcontrol(non-inoculatedYEMBmedium).Itis con-ceivablethatoneormoreofthemolecularsignalsmayimpact biofilmphysiology.33,34 _{ExposureofRT1toexogenous}

addi-tionsof3-oxo-C8-HSLor3-OH-C14-HSLhadpositiveeffectson

physiologicalprocessesrelatedtosurvivaland colonization ability,particularlythemotility andbiofilm formation. Rin-audiandGonzalez(2009)35_{exploredtheroleplayedbyAHL}

molecules inbiofilm formation and their importance with respecttosymbioticrelationshipswiththehost.

TheabilityofrhizobiatoproduceAHLsiswellknowntobe speciesandstrain-dependent.6,36_{Thereareseveraldifferent}

typesofQSsignalingmechanismsthatusearangeof differ-entsignaltypesanddetectionmechanisms.37_Oneofthemost

commonofthesemechanismsinvolvesacyl-homoserine lac-tone(AHL)signalmolecules.Furthermore,AHL-basedquorum sensingappearstobehighlyconservedamongbacteria,and morethan50specieshavebeenidentifiedtoproduceAHLs. AHLmoleculesare described asamphipathic because they containbothpolarandnon-polarregions,sincethe homoser-inelactoneringishydrophilicandthefattyacidsidechainis hydrophobic,andthisamphipathicityappearstofacilitatethe freediffusionofAHLswithintheaqueousintra-and extracel-lularenvironments,andacrossthephospholipidbilayer.37

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 1 2 3 4 5 Time (h)

Without AHL molecule With AHL molecule

OD at

600

nm

10 15 20 24

Fig.5–GrowthcurveofR.etliRT1straininTSBbrothwith orwithouttheadditionof3-oxo-C8-HSL(5␮M).Valuesare means±SEMsofexperimentsperformedinquadruplicate (p<0.05).

Inadditiontocellsignaling,AHLscarryinglongacyl-side chainspossesssignificantsurfaceactivitiesatbiologically rel-evantconcentrations.24_{Generallyswarmingmotilityislinked}

with biofilm formation, although the relationship between these phenomenaappearstoberatherdual.14 _Attachment

is pre-requisite ofbiofilm formation as it governs interac-tionsbetweenbacterialandplanttissues,andhasbeenshown torelyonquorumsensing.Venturi(2006)reportedthatthe extentofswarmingdependsonthestructureofbiofilms dur-ingtheearlystageofformation.38

Bacterialgrowthanalysis

As shown in Fig. 5, the growth rate ofRT1 was enhanced slightlybythepresenceof3-oxo-C8-HSLat5␮M.Furthermore,

co-inoculationofRT1with3-oxo-C8-HSLcausednosignificant

growthdifference.Overall,ourfindingssuggestthat3-oxo-C8

-HSLneitherpromotesnorinhibitsbacterialgrowth.Increased celldensity(exponentialphase)favorsthereleaseofchemical signalsforsocialinteractionsinbiofilms,whichaddsanother level ofcomplexity.39 _{Idealapproachuse toconfirmthat a}

moleculeisaquorumsensingmoleculeistoconfirmthatthe moleculehasnoimpactonfinaldensityafterasuitable incu-bationperiodandthatithasnoeffectongrowthkineticsat definedconcentrations.40

Conclusions

Thisstudy showsendophyticRT1fromthe rootnodulesof lentilsproduceshighlevelsofAHLsandthattheseregulate QScontrolofswarmingmotilityandbiofilmformation.Based onourfindings,weconcludethatquorumsensing,motility and biofilmformationobservedinvitroareaffected byAHL moleculetypeandconcentration,andthattheoverall behav-ioroflentil-nodulatingsoilrhizobialpopulationsissubjected to the fine and coordinated regulationof the synthesis of AHLsinresponsetospecificenvironmentalfactors.Further

studies are in progressto determine the putative quorum sensingmechanismofRT1.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Oneofus(SwarnitaDixit)isthankfultotheChiefExecutive Officer,BiotechPark,Lucknow(India)forprovidinglaboratory facilities.

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