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RESEARCHARTICLEOpenAccessThedistinctivecelldivisioninteractomeofNeisseriagonorrhoeaeYinanZou1,2,YanLi2,3andJo-AnneR.
Dillon1,2,3*AbstractBackground:BacterialcelldivisionisanessentialprocessdrivenbytheformationofaZ-ringstructure,asacytoskeletalscaffoldatthemid-cell,followedbytherecruitmentofvariousproteinswhichformthedivisome.
Thecelldivisioninteractomereflectsthecomplementofdifferentinteractionsbetweenalldivisomeproteins.
Todate,onlytwocelldivisioninteractomeshavebeencharacterized,inEscherichiacoliandinStreptococcuspneumoniae.
ThecelldivisonproteinsencodedbyNeisseriagonorrhoeaeincludeFtsZ,FtsA,ZipA,FtsK,FtsQ,FtsI,FtsW,andFtsN.
ThepurposeofthepresentstudywastocharacterizethecelldivisioninteractomeofN.
gonorrhoeaeusingseveraldifferentmethodstoidentifyprotein-proteininteractions.
WealsocharacterizedthespecificsubdomainsofFtsAimplicatedininteractionswithFtsZ,FtsQ,FtsNandFtsW.
Results:Usingacombinationofbacterialtwo-hybrid(B2H),glutathioneS-transferase(GST)pull-downassays,andsurfaceplasmonresonance(SPR),nineinteractionswereobservedamongtheeightgonococcalcelldivisionproteinstested.
ZipAdidnotinteractwithanyothercelldivisionproteins.
ComparisonsoftheN.
gonorrhoeaecelldivisioninteractomewiththepublishedinteractomesfromE.
coliandS.
pneumoniaeindicatedthatFtsA-FtsZandFtsZ-FtsKinteractionswerecommontoallthreespecies.
FtsA-FtsWandFtsK-FtsNinteractionswereonlypresentinN.
gonorrhoeae.
The2Aand2BsubdomainsofFtsANgwereinvolvedininteractionswithFtsQ,FtsZ,andFtsN,andthe2AsubdomainwasinvolvedininteractionwithFtsW.
Conclusions:ResultsfromthisresearchindicatethatN.
gonorrhoeaehasadistinctivecelldivisioninteractomeascomparedwithothermicroorganisms.
Keywords:Celldivision,Interactome,N.
gonorrhoeae,Protein-proteininteraction,Bacterialtwo-hybridassay,Surfaceplasmonresonance,GSTpull-down,FtsAdomainsBackgroundCelldivisionisessentialforbacterialsurvival.
InEscheri-chiacoli(Ec),normalcelldivisionisdrivenbythefor-mationofanFtsZ-ringatthedivisionsite[1],followedbytherecruitmentofotheressentialproteins,whichtogetherformthedivisome[2].
Genesencodingmostcelldivisionproteinsarelocatedinaconservedregion,thedivisionandcellwall(dcw)cluster[3].
dcwclustershavebeenidentifiedinmostbacterialspecies,includingE.
coli,Bacillussubtilis(Bs),Streptococcuspneumoniae(Sp),Caulobactercrescentus(Cc)andNeisseriagonorrhoeae(Ng)[4–7].
Althoughthegeneorganizationofthedcwclustervariesindifferentbacteriaspecies[8],proteinsinvolvedinthecelldivisionprocessarerelativelyconserved[9,10].
E.
coliencodestenessentialcelldivisionproteins,includingFtsZ,FtsA,ZipA,FtsK,FtsQ,FtsB,FtsL,FtsW,FtsI,andFtsN[11,12].
AssemblyoftheFtsZ-ringstructureisinitiatedwiththepolymerizationofFtsZ,drivenbyGTPhydrolysis,atthemid-cell[13].
FtsAandZipAarerecruitedbyFtsZandanchorFtsZtotheinnermembrane[14].
AftertherecruitmentofFtsK,aDNAtranslocaseinvolvedinDNAsegregation[15–17],theproteincomplexesFtsQ-FtsB-FtsLandFtsW-FtsIarelocalizedtotheseptalring,sequentially[15,18].
RecentstudiesshowedthattheFtsQ-FtsB-FtsLcomplexservesasasignalsensorwhichpromotescellwallremodelingne-cessaryforcellconstriction[19].
FtsIisahigh-molecular-*Correspondence:j.
dillon@usask.
ca1DepartmentofMicrobiologyandImmunology,CollegeofMedicine,Saskatoon,SKS7N5E5,Canada2VaccineandInfectiousDiseaseOrganization,InternationalVaccineCentre,Saskatoon,SKS7N5E3,CanadaFulllistofauthorinformationisavailableattheendofthearticleTheAuthor(s).
2017OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Zouetal.
BMCMicrobiology(2017)17:232DOI10.
1186/s12866-017-1140-1weighttranspeptidasethatcross-linksglycanstrands.
TheFtsW-FtsIcomplexispartofthepeptidoglycansynthesismachinery,andFtsW,alipidIIflippase,transportsthecellwallprecursoracrossthemembrane[20,21].
FtsNisre-cruitedasthelastessentialdivisionproteinthatinitiatescellconstriction[22].
Usingabacterialtwo-hybrid(B2H)assay,anE.
colicelldivisionprotein-proteininteractionnetwork,thecelldivisioninteractome,whichincluded16interactionsbe-tween10celldivisonproteins,wasidentified[23,24].
ThecelldivisioninteractomeofS.
pneumoniaewasalsocharacterizedusingacombinationofB2Handco-immunoprecipitationassays[25].
Atotalof17interac-tionswasobservedamongninecelldivisionproteinsofS.
pneumoniaewhichincludedFtsZ,FtsA,FtsK,DivlB,DivlC,FtsL,FtsW,andPBP2x[25].
Todate,E.
coliandS.
pneumoniaearetheonlytwoorganismswithcharac-terizedcelldivisioninteractomes[23–25].
N.
gonorrhoeaeisaGram-negativediplococcusthatcausesgonorrheainhumans[26].
PreviousstudiesonN.
gonorrhoeaecelldivisionfocusedonitsMinsystemwhichlocalizesFtsZtothemid-cell,andFtsZ[27–29].
N.
gonorrhoeaealsocontainsadcwclusterwhichen-codes5celldivisionproteins-FtsZ,FtsA,FtsQ,FtsW,andFtsI[7].
Othernon-dcwclusterdivisomeproteinsencodedbyN.
gonorrhoeaeincludeZipA,FtsK,andFtsN.
AscomparedtoE.
coli,N.
gonorrhoeaelacksFtsBandFtsL[7].
ToinvestigatethecelldivisioninteractomeinN.
gonor-rhoeae,itscelldivisionproteininteractionswereidentifiedusingacombinationofB2HandglutathioneS-transferase(GST)pull-downassays,aswellassurfaceplasmonreson-ance(SPR).
Weidentifiednineinteractionsamongtheeightcelldivisionproteinstested.
WealsoidentifiedthesubdomainsofFtsANginvolvedinitsinteractionwithFtsQNg,FtsZNg,FtsNNg,andFtsWNg.
ComparisonofthecelldivisioninteractomesofE.
coli,S.
pneumoniaeandN.
gonorrhoeaeindicatesthatN.
gonorrhoeaepossessesadistinctivecelldivisioninteractome.
MethodsStrainsandgrowthconditionsThebacterialstrainsandplasmidsusedinthisstudyareshowninTable1.
E.
coliDH5αandXL1-Bluewereusedashostsforcloning.
E.
coliBL21(DE3)andC41(DE3)wereusedashostsforproteinpurification.
E.
coliR721wasusedinB2Hassays[30].
E.
coliDH5α,XL1-Blue,BL21(DE3)andC41(DE3)weregrowninLuria-Bertani(LB)medium(BDDifco,Sparks,MD),for16–18h(hr),at37°C.
E.
coliR721wasgrownunderthesamecondi-tionsandincubatedat34°C,asdescribedpreviously[24].
N.
gonorrhoeaeCH811wasgrownonGCmediumbaseagar(GCMB,Oakville,ON),supplementedwithKellogg'sdefinedsupplement(GCMBK,40gD-glucose,1gglutamine,10mlof0.
5%ferricnitrateand1mlof20%cocarboxylase),at35°C,inahumidenvironment,with5%CO2,for18to24h[31].
Whenrequired,thefollowingconcentrationsofantibi-oticswereaddedtoLBmedium:100μg/mlampicillin(Sigma,Oakville,ON)or50μg/mlkanamycin(Sigma).
ForB2Hassays,34μg/mlchloramphenicol(Sigma),30μg/mlkanamycin,and50μg/mlampicillinwereaddedtoLBmedium.
DNAmanipulationsN.
gonorrhoeaeCH811genomicDNAwaspurifiedusingaQIAampgenomicDNAkit(Qiagen,Mississauga,Ontario,Canada).
DNAsampleswerestoredat20°C.
Oligonucleotidesforpolymerasechainreaction(PCR)amplificationsweresynthesizedbyInvitrogen(Table2;Burlington,Ontario,Canada).
PCRswereperformedinaGeneAmpPCRsystem9700(AppliedBiosystems,Fos-terCity,CA,USA)asfollows:4min(min)at94°C,30cyclesofdenaturationfor1minat94°C,annealingfor45s(s)at55°C,extensionfor1.
5minsat72°C,and10minsat72°C.
PCRswerecarriedoutin100-μl(finalvolume)mixturescomprising71.
5μldouble-distilledH2O(ddH2O),10μlof10*PCRbuffer[15mMMgCl2,4μlof10mMdeoxynucleosidetriphosphate(dNTP),2μlofeachprimer(0.
2μg/ml),0.
5μlofTaqDNApoly-merase(5U/μl;NewEnglandBioLabs,Ontario,Canada)],and,10μlofpurifiedN.
gonorrhoeaeCH811genomicDNAsuspension.
Bacterialtwo-hybridassaysThemethoddevelopedbyDiLalloetal.
[24]wasusedforallB2Hassays.
ftsA,ftsK,ftsQ,ftsI,ftsW,andftsNwereamplifiedfromN.
gonorrhoeaeCH811byPCRusingtheprimerpairsP1/P2,P3/P4,P5/P6,P7/P8,P9/P10,andP11/P12(Table2),respectively.
PCRampliconsweredigestedwithBamHIandSalIandligatedintopre-viouslydigestedpcIp22andpcI434vectors,toproducepclp22-A,pcIp22-K,pcIp22-I,pcIp22-W,pcIp22-Q,pcIp22-N,pcI434-A,pcI434-K,pcI434-I,pcI434-W,pcI434-Q,andpcI434-N(Table3).
zipANgwasamplifiedfromN.
gonor-rhoeaeCH811genomicDNAusingtheprimerpairP13/P14(Table2);thePCRampliconswasdigestedwithBglIIandBamHI,andligatedintopre-digestedpcIp22andpcI434toproducepcIp22-ZipAandpcI434-ZipA.
pcIp22-ZandpcI434-Zconstructsweregeneratedpreviously[32].
TheexpressionofftsANg,ftsZNgandzipANgfromB2HconstructswasverifiedbyWesternblotanalysisusingappropriateantibodiespreparedinourlabusingprevi-ouslydescribedmethods[33].
Theseproteinswereexpressedfromthevectorsundertheconditionstested(datanotshown).
Theexpressionoftheseproteinsindi-catedthatanynegativeB2Hinteractionsinvolvingthemwasnotafunctionoflackofexpression.
Zouetal.
BMCMicrobiology(2017)17:232Page2of14ToascertainwhatsubdomainsofFtsANginteractedwithgonococcalcelldivisionproteinsFtsZNg,FtsQNg,FtsWNg,orFtsNNg,sixpreviouslycreatedtruncationsoftheprotein(T1,T2,T3,T4,T5,andT6;Additionalfile1:FigureS1)wereused[33].
PlasmidconstructsforB2Hassayswerepreviouslygenerated[33].
B2Hassayswereperformedasdescribedpreviously[24].
Thisassayisbasedonthereconstitutionofachimericrepressorthatbindstothe434/P22hybridoperatorandrepressestheexpressionofadown-streamlacZgeneinE.
coliR721.
EachgenetestedforapotentialinteractionwasclonedintopcIp22andpcI434andrecombinantconstructsweretransformedintoE.
coliR721eithersinglyorincombination.
N.
gonorrhoeaeFtsZself-interactionwasusedaspositivecontrol.
R721withoutplasmidsandsingleplasmidtransformantswereusedasnegativecontrols.
R721withoutplasmidshadaβ-galactosidaseactivityof2504±34Millerunits.
Theβ-galactosidaseactivityofeachcombinationwascomparedtothatofR721.
Valuesoflessthan50%(1250MillerUnits)indi-cateanegativeinteraction[24].
StatisticalanalyseswereperformedusingtheunpairedStudentt-test.
StandarddeviationsweredeterminedforthemeanvalueofMillerunitswherethreeindependentexperi-mentswereperformed.
Table2PrimersdesignedinthisstudyPrimernameSequences(5′-3′)P1FtsA-reBamHIGCGCGGATCCTCAGAGGTTGTTTTCAATCCP2FtsA-fwSalIGCGCGTCGACCATGGAACAGCAGAAAAGATACP3fwSalI-ftsKGCGCGTCGACCATGTTTTGGATAGTTTTGATCGTTATP4reBamHI-ftsKCGCGGGATCCTCAAGCATTGTCCAAGGGGACGAGP5fwSalI-ftsQGCGCGTCGACCATGTGGGATAATGCCGAAGCGATGP6reBamHI-ftsQCGCGGGATCCCTATTCTTCGGATTCTTTTTCGGGP7fwSalI-ftsIGCGCGTCGACCATGTTGATTAAAAGCGAATATAAGCCP8reBamHI-ftsICGCGGGATCCTTAAGACGGTGTTTTGACGGCTGCP9fwSalI-ftsWGCGCGTCGACCATGAAGATTTCGGAAGTATTGGTAAAP10reBamHI-ftsWCGCGGGATCCTTACTCCACCCGGTAACCGCGCATP11fwSalI-ftsNGCGCGTCGACCATGTTTATGAACAAATTTTCCCAATCP12reBamHI-ftsNCGCGGGATCCTTATTTGCCTTCAATCGCACGGATP13fwBglII-ZipAGCGCGAGATCTGATGATTTACATCGTACTGTTCCTCP14reBamHI-ZipACGCGGGATCCTTATGAAAACAGGCGCAGGGCP15FtsA-reEcoRI-pET30aATATCGAATTCTCAGAGGTTGTTTTCAATCCACCP16FtsA-fwBglII-pET30aAGCCCAGATCTGATGGAACAGCAGAAAAGATACATCP17fwBglII-FtsQ-pET30aAGCCCAGATCTGATGTGGGATAATGCCGAAGCGATGP18reEcoRI-ftsQ-pET30aATATCGAATTCCTATTCTTCGGATTCTTTTTCGGGP19FtsZ-fwBglII-pET30aAGCCCAGATCTGATGGAATTTGTTTACGACGTGGCAP20FtsZ-ReEcoRI-pET30aAGCCCGAATTCTTATTTGTCTGAATTGTGTTGACGP21fwFtsA-BamHI-GSTCGCGGGATCCATGGAACAGCAGAAAAGATACATCP22fwEcoRI-FtsNGACGAATTCATGTTTATGAACAAATTTTCCCAATCCP23reXhoI-FtsNGACCTCGAGTTATTTGCCTTCAATCGCACGTable1BacterialstrainsusedinthisstudyStainRelevantcharacteristicsSource/referenceE.
coliDH5αsupE44ΔlacU169(80lacZΔM15)hsdR17endA1gyrA96thi-1relA1GibcoE.
coliXL1-BluerecA1endA1gyrA96thi-1hsdR17supE44relA1lac[F′proABlaclqZΔM15]Tn10StratageneE.
coliBL21(DE3)F,dcmΔ,ompT,hsdS(rBmB),gal,λ(DE3)StratageneE.
coliC41(DE3)FompThsdSB(rBmB)galdcm(srl-recA)306::Tn10(Tetr)(DE3)[70]E.
coliR721supEthyD(lac-proAB)F′[proAB+lacIqlacZDM15]glpT::O-P434/P22lacZ[30]N.
gonorrhoeaeCH811Auxotype(A)/serotype(S)/plasmidcontent(P)class:nonrequiring/IB-2/plasmid-free,Strr[71]Zouetal.
BMCMicrobiology(2017)17:232Page3of14Constructionandpurificationofhis-fusionproteinsForHis-fusionconstructs,full-lengthftsA,ftsQ,andftsZwerePCR-amplifiedfromN.
gonorrhoeaeCH811genomicDNAusingprimerpairsP15/P16,P17/P18andP19/P20(Table2),respectively.
PCRampliconsweredigestedwithEcoRIandBglIIandligatedintopre-digestedpET30a,tocreatepETA,pETQ,andpETZ.
PlasmidpETAwastrans-formedintoE.
coliC41(DE3)andplasmidspETQandpETZweretransformedintoE.
coliBL21(DE3).
Theover-expressionofallfusionproteinswasinducedwith400μMIPTG,at30°C,for2h.
PurificationofHis-FtsANg,His-FtsZNg,andHis-FtsQNgwascompletedusingHisBindResin(EMDMillipore,Billerica,MA),followingthemanu-facturer'sinstructions.
His-FtsZNgwasfurthertreatedwiththrombinprotease(EMDMillipore,Billerica,MA),over-night,at4°C,tocleavetheN-terminalHistag.
Thrombinwasremovedusing100μlofp-aminobenzamidine-agarose(Sigma#A7155).
FtsZwasdialyzedagainstMESbuffer(50mMMES,300mMKCl,10mMMgCl2,pH7.
5)priortouseinFtsZpolymerizationexperiments[34].
Table3PlasmidsusedinthisstudyPlasmidRelevantgenotypeSource/ReferencepcIp22pC132derivativecarryingN-terminalendofP22repressor[30]pcI434pACYC177derivativecarryingN-terminalendof434repressor[30]pcIp22-ApcIp22derivativecarryingtheftsANggeneThisstudypcI434-ApcI434derivativecarryingtheftsANggeneThisstudypcIp22-KpcIp22derivativecarryingtheftsKNggeneThisstudypcI434-KpcI434derivativecarryingtheftsKNggeneThisstudypcIp22-QpcIp22derivativecarryingtheftsQNggeneThisstudypcI434-QpcI434derivativecarryingtheftsQNggeneThisstudypcIp22-IpcIp22derivativecarryingtheftsINggeneThisstudypcI434-IpcI434derivativecarryingtheftsINggeneThisstudypcIp22-WpcIp22derivativecarryingtheftsWNggeneThisstudypcI434-WpcI434derivativecarryingtheftsWNggeneThisstudypcIp22-NpcIp22derivativecarryingtheftsNNggeneThisstudypcI434-NpcI434derivativecarryingtheftsNNggeneThisstudypcIp22-ZpcIp22derivativecarryingtheftsZNggene[32]pcI434-ZpcI434derivativecarryingtheftsZNggene[32]pcIp22-AT1pcIp22derivativecarryingtheftsANggenefragmentencodingaminoacids1–162[33]pcIp22-AT2pcIp22derivativecarryingtheftsANggenefragmentencodingaminoacids1–194[33]pcIp22-AT3pcIp22derivativecarryingtheftsANggenefragmentencodingaminoacids1–230[33]pcIp22-AT4pcIp22derivativecarryingtheftsANggenefragmentencodingaminoacids231–301[33]pcIp22-AT5pcIp22derivativecarryingtheftsANggenefragmentencodingaminoacids302–414[33]pcIp22-AT6pcIp22derivativecarryingtheftsANggenefragmentencodingaminoacids351–414[33]pcI434-AT1pcI434derivativecarryingtheftsANggenefragmentencodingaminoacids1–162[33]pcI434-AT2pcI434derivativecarryingtheftsANggenefragmentencodingaminoacids1–194[33]pcI434-AT3pcI434derivativecarryingtheftsANggenefragmentencodingaminoacids1–230[33]pcI434-AT4pcI434derivativecarryingtheftsANggenefragmentencodingaminoacids231–301[33]pcI434-AT5pcI434derivativecarryingtheftsANggenefragmentencodingaminoacids302–414[33]pcI434-AT6pcI434derivativecarryingtheftsANggenefragmentencodingaminoacids351–414[33]pET30aKanRPT7::6XhisEMDMillipore,Billerica,MApETApET30aderivativecarryingtheftsANggeneThisstudypETQpET30aderivativecarryingtheftsQNggeneThisstudypETZpET30aderivativecarryingtheftsZNggeneThisstudypGEX2TAmpRPtac::gst::lacIqAmershamBioscience,Uppsala,SwedenpGEXApGEX2TderivativecarryingtheftsANggeneThisstudypGEXNpGEX2TderivativecarryingtheftsANggeneThisstudyZouetal.
BMCMicrobiology(2017)17:232Page4of14GSTpull-downassayForGSTfusionconstructs,full-lengthftsAandftsNwerePCR-amplified,fromN.
gonorrhoeaeCH811,usingpri-merpairsP21/P18andP22/P23(Table2),respectively.
TheftsAampliconwasdigestedwithBamHIandEcoRIandligatedintopre-digestedpGEX2T,tocreatepGEXA(Table3).
TheftsNampliconwasdigestedwithEcoRIandXhoIandligatedintopre-digestedpGEX2T,produ-cingpGEXN(Table3).
PlasmidspGEXAandpGEXNweretransformedintoE.
coliC41(DE3)andE.
coliBL21(DE),respectively.
OverexpressionofGST-FtsAandGST-FtsNwasaccomplishedbyinductionwitheither400μMor800μMofIPTG,respectively,at30°C,for2h.
PurificationofGST-FtsAandGST-FtsNwascar-riedoutusingGSTBindResin(EMDMillipore,Billerica,MA),followingthemanufacturer'sinstructions.
PurifiedGST-fusionandHis-fusionproteinswereincubatedwithpre-equilibratedGSTBindResininphosphatebufferedsaline(PBS)buffer(137mMNaCl,2.
7mMKCl,10mMNa2HPO4,1.
8mMKH2PO4,0.
5%Triton-X100,1mMDTT,pH7.
9)at4°Covernight.
Pre-purifiedGSTwasusedasanegativecontrol.
Thepre-boundresinwascollectedbycentrifugationandwashedinPBSthreetimes.
Boundproteinsweredissoci-atedfromresinbyadding5XLaemmlibuffer,separatedbyelectrophoresison10%sodiumdodecylsulfatepoly-acrylamidegels(SDS-PAGE),andidentifiedbyWesternblotusingpolyclonalanti-GSToranti-6*Hisantibodies(ThermoScientific;Waltham,MA),sequentially.
ForFtsANg-FtsZNginteractions,theGSTpull-downassaywasperformedinMESbuffer(50mMMES-NaOH,50mMKCl,10mMMgCl2,0.
5%Triton-X100,1mMATP,2mMGTP,pH7.
5)[34].
TopromotethepolymerizationofFtsZNgnecessaryforthisinteraction,FtsZNgwastreatedwith2mMGTPand1mMATP,asdescribedpreviously[34],beforemixingwithGST-FtsANgandGSTBindResin.
AllGSTpull-downassayswereperformedminimallyinduplicate.
FtsZpolymerizationassaysFtsZNgpolymerizationwasmeasuredby90°anglelightscatteringusingaDynapro-MS800instrument(WyattTechnologyCorporation)withawavelengthof310nmandaslitwidthof0.
5mm.
MESbufferisoptimalforFtsZpolymerizationwhichisrequiredtoobserveanFtsA-FtsZinteraction[34,35].
FtsZNg(~6μM)inMESbuffer(50mMMES-NaOH,50mMKCl,10mMMgCl2,pH7.
5)wasinjectedintoa45ulquartzcuvetteandwarmedto30°C,priortothemeasurement.
Datawerecollected,for4min,fromunpolymerizedFtsZNgtoestablishabaseline.
GTPwasthenaddedtoafinalconcentrationof2mManddatawerecollectedevery5sfor25min.
Datawerere-cordedandanalyzedusingDynamicsv5software.
NegativestainelectronmicroscopywasusedtovisualizeFtsZNgpolymers.
5μlofFtsZ(6μM)with,orwithout,GTP(finalconcentration2mM)wasincubated,at30°C,for5min.
Themixturewasplacedonacarbon-coatedcoppergrid(400meshsize)for2minandthenblotdried.
ThegridcontainingFtsZNgwasstainedwith1%uranylacetate,blotted,andair-driedfor3h.
PolymerswerevisualizedandphotographedusingaHitachitransmissionelectronmicroscopyHT7700.
Surfaceplasmonresonance(SPR)ProteininteractionswereexaminedbySPRusingaBio-RadXPR36(Bio-RadLaboratories)instrumentandaProteOnHTESensorChip(Bio-RadLaboratories).
Thechipsurfacewasregeneratedbyinjectionof0.
5%SDS,50mMNaOH,100mMHCland300mMEDTA,ataflowrateof30μl/min,for120s.
Activationwasper-formedusing500μMofNiSO4.
ForFtsANg-FtsNNgandFtsANg-FtsQNgSPRexperi-ments,ligands(i.
e.
His-FtsNNgforFtsANg-FtsNNg,andHis-FtsQNgforFtsANg-FtsQNginteractions)wereimmobi-lizedontothesensorchipataconcentrationof200nM.
Atwo-folddilutionseriesoftheanalyte(FtsANg),inPBSbufferwithTween-20(PBST;137mMNaCl,2.
7mMKCl,10mMNa2HPO4,1.
8mMKH2PO4,0.
1%BSA,0.
05%Tween-20,pH7.
9),wasinjectedataflowrateof30μl/minoverthesurfaceofthechipfor120s.
ThiswasfollowedbyaninjectionofPBSTbufferfor300s.
NegativecontrolscomprisedareferencechannelflowedwithPBSTbuffer,andachipsurfaceimmobilizedwitheitherFtsQNgorFtsNNgflowedwithGSTinPBST.
FortheFtsANg-FtsZNginteraction,theSPRbindingassaywasperformedusingMESbuffer,supplementedwith0.
1%BSA,0.
05%Tween-20,and1mMATPwereaddedwiththepHadjustedto7.
5.
FtsANgwasimmobilizedonthechipsurfaceasdescribedabove.
Each120-sinjectionofpolymerizedFtsZNgwasfollowedbyaninjectionofsup-plementedMESbufferfor300sfordissociation.
NegativecontrolsincludedareferencechannelwhichwasflowedwithMESbuffercontaining2mMGTP,andtheFtsANg-immobilizedchipsurfaceflowedwithGSTinsupple-mentedMESinsteadofpolymerizedFtsZNg.
AllSPRdatawasanalyzedusingProteOnManager(Bio-RadLaboratories).
Thesensorgram(i.
e.
agraphoftheresponseunitversustime)wasfirstsubtractedbytheresponseunits(RU)ofthereferencechannel,withnoimmobilizedligands,toreducethenon-specificbind-ingsignalsbetweenanalyteandemptychipsurface.
Then,thesensorgramwassubtractedwiththeRUsignalwithrunningbufferandligandimmobilizedonthechip.
AssociationanddisassociationconstantswereobtainedusingtheLangmuir1:1kineticfitmodel,bynonlinearregression,usingProteOnManager.
Eachproteinpairwastestedminimallyinduplicate.
Zouetal.
BMCMicrobiology(2017)17:232Page5of14ResultsIdentificationofN.
gonorrhoeaecelldivisionproteininteractionsbybacterialtwo-hybridassayUsingB2Hassays,weinvestigated28potentialinterac-tionsamongeightgonococcaldivisomeproteinsinclud-ingFtsZ,FtsA,ZipA,FtsK,FtsQ,FtsI,FtsW,andFtsN.
Theresults(Table4)showthatnineinteractions,FtsZ-FtsA,FtsZ-FtsK,FtsZ-FtsW,FtsA-FtsK,FtsA-FtsQ,FtsA-FtsW,FtsA-FtsN,FtsI-FtsW,andFtsK-FtsN,dis-playedaresidualβ-galactosidaseactivitylowerthan50%,indicatingapositiveinteractionbetweentheseproteinsinN.
gonorrhoeae.
TheinteractionbetweenFtsANgandFtsNNghadthelowestresidualβ-galactosidaseactivity(24%),indicatingthestrongestinteraction.
ThiswasfollowedbyFtsANg-FtsKNg(30%),FtsNNg-FtsKNg(31%),FtsINg-FtsWNg(35%),FtsZNg-FtsWNg(39%),FtsANg-FtsZNg(40%),FtsZNg-FtsKNg(41%),FtsANg-FtsWNg(45%),andFtsANg-FtsQNg(48%)interactions.
ZipANgdidnotdirectlyinteractwithothercelldivisionproteinsastheresidualβ-galactosidaseactivityofallinteractionswasabove50%(Table4).
GSTpull-downofFtsANg-FtsQNg,FtsANg-FtsZNgandFtsANg-FtsNNginteractionsToconfirmtheresultsofselectedB2Hassays,weexaminedseveralinteractions(i.
e.
FtsQNg-FtsANg,FtsANg-FtsNNg,FtsANg-FtsZNg)usingGSTpull-downassays.
GSTpull-downresults(Fig.
1a)showedthatHis-FtsQNgwaspulleddownbyGST-FtsANg,butnotGSTitself(negativecontrol),indicatinganinteractionbetweenFtsANgandFtsQNg.
Usingsimilarevaluationcriteria,weascertainedthatHis-FtsANgwaspulleddownbyGST-FtsNNg,indicatinganinteractionbetweenthesetwoproteins(Fig.
1b).
TheinteractionsofFtsANgandFtsZNgfromE.
coliinvitrorequiresthepresenceofbothATPandGTP[35].
GTPpromotesFtsZpolymerization,andATPisneces-saryforFtsAtointeractwithFtsZ,butnotforFtsZpolymerization[36,37].
ThepresenceofFtsZNgpoly-mersinMESbufferwasdeterminedbytransmissionelectronmicroscopy(TEM)anddynamiclightscattering(DLS;Additionalfile2:FigureS2).
GSTpull-downassaydidnotdetectaninteractionbetweenFtsANgandFtsZNginthepresenceof1mMATPand2mMGTP(Fig.
1c).
Thisresultwasunexpected,givenourB2HresultsandthecommonalityofFtsA-FtsZinteractioninotherbac-terialspecies[24,25,38,39],asascertainedbydifferentinvivoassays(i.
e.
B2H,yeasttwo-hybrid,chemicalcross-linkingwithco-immunoprecipitation).
SurfaceplasmonresonanceevaluationofFtsANg-FtsQNg,FtsANg-FtsZNgandFtsANg-FtsNNginteractionsSurfaceplasmonresonance(SPR)wasusedtoconfirmselectedgonococcalcelldivisionprotein-proteininterac-tionsinreal-time.
SPRwasusedtoevaluatetheinterac-tionsofFtsANgwithFtsZNgbecauseoftheconflictingresultsobservedwithB2HandGSTpull-downassays.
GTPwasaddedtopromoteFtsZNgpolymerization(Additionalfile2:FigureS2).
ThesensorgramindicatedthatFtsZNginteractedwithFtsANgatconcentrationsof6μMand12μM(Fig.
2a),butnotatconcentrationslowerthan6μM(datanotshown).
KineticanalysisshowedthattheFtsANg-FtsZNginteractionhadaslowassociation(ka=3.
56*102M1s1)andasignificantTable4InteractionsbetweeneightcelldivisionproteinsinN.
gonorrhoeaeasdeterminedbyB2HassayBycomparisontopositivecontrols(E.
coliR721withoutplasmids),interactionswithlessthan50%ofresidual-galactosidaseactivity(framed)wereconsideredaspositive.
FtsZNgself-interactionwasusedasapositivecontrol.
Thenumbersrepresentpercentageofmean-galactosidaseactivity,±standarddeviation*Statisticallysignificant(P≤0.
05);NS:notstatisticallysignificant(P>0.
05)Zouetal.
BMCMicrobiology(2017)17:232Page6of14disassociationactivity(kd=5.
31*103s1),givingaKDvalueof14.
9μM.
Thissuggestedthattheinteractionbe-tweenFtsANgandFtsZNgwaslikelytransient.
WhenGTPwasabsentfromtheFtsZNgproteinsolution,nobindingwasdetectedbetweenFtsANgandFtsZNg(datanotshown).
ThesensorgramoftheinteractionbetweenFtsANgandthenegativecontrol(GST)alsoshowednobindingactivity(Fig.
2b),indicatingthespecificityoftheSPRresultsfortheinteractionofFtsANgwithFtsZNg.
FortheSPRanalysisoftheFtsANg-FtsQNginteraction,FtsANgwastestedusingvariousconcentrations(from31.
25nMto250nM;Fig.
2c).
At0s,theassociationofFtsANgandFtsQNgwasobservedimmediatelyfollowinginjectionoftheFtsANgsolutionontotheFtsQNg-la-beledchipsurface,witharapidincreaseofresponseunits(ka=2.
72*105M1s1;Fig.
2c).
Thisindicatedafastbindingeventbetweenthetwoproteins.
Disas-sociationbetweenFtsANgandFtsQNgwasnotsignifi-cant(kd=4.
09*103s1),suggestingthisinteractionwasstrongandstable(KD=15.
1nM).
Thenegativecontrol,usingnon-interactingGST,didnotcauseanychangeintheresponseunits(Fig.
2d).
TheFtsANg-FtsNNginteractionwasobservedwithanincreasingconcentrationofFtsANg(62.
5nM,125nM,250nMand500nM;Fig.
2e).
His-FtsNNghadabindingaffinity(KD)of53.
3nMwithFtsANg.
Theassociationanddisassociationconstantswere1.
15*105M1s1,and6.
16*103s1,respectively(Fig.
2e),indicatingastronginteractionbetweenFtsANgandFtsNNg.
Theinjectionofnon-interactingGSTontotheFtsNNgimmo-bilizedchipsurfacedidnotcauseanychangeintheresponseunits(Fig.
2f).
The2Aand2BsubdomainsofFtsANginteractswithFtsZNg,FtsNNg,FtsWNgandFtsQNgSinceFtsANginteractedwithFtsZNg,FtsQNg,FtsWNg,andFtsNNg,wefurtherexaminedtheinteractionregionsofFtsANgwiththesefourproteinsusingB2Hassays.
BasedonFtsANghomologymodeling,sixFtsANgtrunca-tions(T1-T6)werecreated(Additionalfile1:FigureS1),whichcontainedoneormoreFtsANgsubdomains[33].
FtsZNgself-interactionwasusedasapositivecontrol.
AndnegativecontrolsincludedE.
coliR721withoutplasmidsorcarryingeachsinglerecombinantB2Hvectorinwhichthegeneofinteresthadbeencloned.
FtsANgtruncationsT3,T4,andT5interactedwithFtsZNgandFtsNNg(Figs.
3and4,bluebars).
FtsANgtruncationsT1,T2,andT6didnotshowaninteractionwiththeseproteins(Figs.
3and4,greenbars).
TheT4andT5truncationsincludedthe2Band2A2subdo-mainsofFtsANg,suggestingthatthesesubdomainsofFtsANginteractedwithbothFtsZNgandFtsNNg.
TheT3constructcontainedalsocontainedthe2A1subdomainofFtsANg,ascomparedtotruncationsT1andT2,indi-catingthatthissubdomainwasalsoinvolvedininterac-tionswithFtsZNgandFtsNNg.
FtsQNginteractedonlyFig.
1InteractionsofFtsANgwithFtsQNg,FtsNNgandFtsZNgbyGSTpull-down.
aGSTpulldownbetweenHis-FtsQNgandGST-FtsANg.
Lane1:His-FtsQNginput;Lane2:GST-FtsANgandHis-FtsQNgmixture;Lane3:GSTandHis-FtsQNgmixture;bGSTpulldownbetweenHis-FtsANgandGST-FtsNNg.
Lane1:His-FtsANginput;Lane2:GST-FtsNNgandHis-FtsANgmixture,GST-FtsNNgwasloadedwithGSTandGST-FtsNNgdegradationproducts;Lane3:GSTandHis-FtsANgmixture;cGSTpulldownbetweenHis-FtsZNgandGST-FtsANg.
Lane1:His-FtsZNginput;Lane2:GST-FtsANgandHis-FtsZNgmixture;Lane3:GSTandHis-FtsZNgmixture;His-taggedfusionproteinswerevisualizedusinganti-6*Hisantibody;GSTandGST-taggedfusionpro-teinswerevisualizedusinganti-GSTantibodyZouetal.
BMCMicrobiology(2017)17:232Page7of14withtheT4andT5truncationsofFtsANg(Fig.
5,bluebars),indicatingthatthe2Band2A2subdomains,butnotthe2A1subdomain,wererequiredfortheFtsANg-FtsQNginteraction.
OnlytheT5truncationofFtsANginteractedwithFtsWNg,suggestingthat2A2subdomainwasinvolvedintheinteractionwithFtsWNg(Additionalfile3:FigureS3).
Insummary,theseresultsshowedthatthe2A1,2A2and2Bsubdo-mainsofFtsANgarerequiredforitsinteractionwithFtsNNgandFtsZNg.
TheFtsANg2A2and2Bsubdo-mainsarerequiredforinteractionwithFtsQNg,andthe2A2subdomainisinvolvedintheinteractionwithFtsWNg.
DiscussionTheN.
gonorrhoeaecelldivisioninteractomedescribedinourstudyisthethirdcelldivisioninteractionnetworkidentifiedinbacteria,inadditiontoE.
coliandS.
pneu-moniae(Fig.
6a)[23–25].
Comparedtotheothertwointeractomes(Fig.
6bandc),fewerinteractionproteinpairsareidentifiedinN.
gonorrhoeae(Fig.
6a).
OnlynineinteractionsarepresentamongtheeightdivisomeFig.
2SPRmeasurementforN.
gonorrhoeaeFtsA-FtsZ,FtsQ-FtsAandFtsA-FtsNinteractions.
a6and12μMofFtsZNgwereanalyzedforinteractionwithFtsANg;bNegativeinteractionbetweenFtsANgandGST;cFtsANgatdifferentconcentrations(31.
25,62.
5,125and250nM)weremeasuredforbindingaffinitytoFtsQNg;dNegativeinteractionbetweenFtsQNgandGST;eFtsNNgatdifferentconcentrations(62.
5,125,250and500nM)wasanalyzedforinteractionwithFtsANg;fNegativeinteractionbetweenFtsNNgandGST.
AssociationanddisassociationconstantswereobtainedusingtheLangmuir1:1kineticfitmodelbynonlinearregressionusingProteOnManager(Bio-RadLaboratories)Zouetal.
BMCMicrobiology(2017)17:232Page8of14Fig.
3InteractionsbetweenFtsANgtruncations(T1,T2,T3,T4,T5andT6)andFtsZNg(Z)byB2Hassays.
R721withoutplasmidsandsingletransformantswereusedasnegativecontrols.
R721withoutplasmidshadaβ-galactosidaseactivityof2504±34Millerunits.
FtsZNgself-interactionwasusedasapositivecontrol.
Valuesoflessthan50%(1250MillerUnites)indicateanegativeinteraction(greenbars)positiveandnegativecontrolsarelabeledinwhite(whitebar)Fig.
4InteractionsbetweenFtsANgtruncations(T2,T3,T4,T5andT6)andFtsNNg(N)byB2Hassays.
Valuesoflessthan50%(1250MillerUnites)indicateanegativeinteraction(greenbars)Zouetal.
BMCMicrobiology(2017)17:232Page9of14Fig.
5InteractionsbetweenFtsANgtruncations(T2,T3,T4,T5andT6)andFtsQNg(Q)byB2Hassays.
Valuesoflessthan50%(1250MillerUnites)indicateanegativeinteractionbetweenthetwoproteins(greenbars)Fig.
6CelldivisioninteractomesofaN.
gonorrhoeae,bE.
coli[23,24],andcS.
pneumoniae[25].
Redlinesindicatecommoninteractions;bluelinesindicateuniqueinteractionsinN.
gonorrhoeaeZouetal.
BMCMicrobiology(2017)17:232Page10of14proteinstestedinN.
gonorrhoeae,whileE.
coliandS.
pneumoniaehave21and17interactionsamongtenandeightdivisomeproteins,respectively[24,25].
ThedevelopmentofallthreecelldivisioninteractomeswasbasedoninteractiondataobtainedfromthesameB2Hsystem[24,25]TheE.
coliinteractomewasdevel-opedusingB2HresultsexclusivelywhiletheS.
pneumo-niaestudyalsoappliedco-immunoprecipitationtoverifyselectedB2Hpositiveinteractionpairs[24,25].
Inourstudy,weusedacombinationofGSTpull-downandsurfaceplasmonresonancetofurtherstudyselectedpositiveB2Hinteractions.
Twointeractions,FtsA-FtsZandFtsZ-FtsK,arecon-servedinthecelldivisioninteractomesofN.
gonor-rhoeae,E.
coliandS.
pneumoniae(Fig.
6,redlines).
TheFtsA-FtsZinteractionisacommoninteractioninpro-karyotes[24,25,39–41].
BothourB2HandSPRresultsconfirmedthisinteractioninN.
gonorrhoeae.
AproperratiobetweenFtsAandFtsZiscrucialfortheinteractioninE.
coli[42]andourSPRresultssupportthisfinding;FtsANginteractswithFtsZNgonlywhenitsconcentra-tionishigherthan6μM(Fig.
3b),indicatingthattheinteractionrequiresacriticalconcentrationthreshold.
OurSPRresultsfurthershowedthatinteractionbetweenFtsANgandFtsZNgwastransient,aresultwarrantingfur-therstudytofullyunderstanditsimplicationsfordivi-someformationinN.
gonorrhoeae.
Unexpectedly,theGSTpull-downassay,aninvitroassay,didnotdetectanFtsANg-FtsZNginteraction.
Webelievethatthis"falsenegative"invitroresultwascausedbytherequirementofamembrane/solidsurfacesupportfortheinteractiontoanchorFtsA[35,43,44].
TheinteractionofFtsZwithFtsKhasbeenobservedinN.
gonorrhoeae,E.
coli,S.
pneumoniae,B.
subtilisandC.
crescentus[24,25,45,46].
TheC-terminusofFtsKisrequiredforproperDNAsegregationinE.
coli[47].
TheabsenceofanFtsZ-FtsKinteractioninbothE.
coliandC.
crescentuscausedabnormalchromosomesegregationandcellfilamentation[45,48].
ThissuggeststhattheFtsZ-FtsKinteractionconnectsthecelldivisionprocesswithchromosomesegregation,byensuringthattherep-licatedchromosomeisclearedfromthedivisionsite.
TheFtsA-FtsWinteractionhasbeenobservedonlyinN.
gonorrhoeae(Fig.
6,bluelines).
SinceFtsWisamem-braneproteinanddifficulttopurify,wedidnotverifytheinteractionbyGSTpull-downandSPRassays.
How-ever,weperformedadditionalB2HassaystoidentifywhichsubdomainsofFtsAwereinvolvedinitsinter-actionwithFtsW(Additionalfile3:FigureS3)andshowedthatthe2A2subdomainofFtsAstronglyinter-actswithFtsW(Additionalfile3:FigureS3).
FtsW,aninnermembraneprotein,isrequiredinE.
coliforthere-cruitmentofFtsIandthetranslocationofthecellwellprecursor,lipidII[20,21,49,50].
AnFtsI-FtsWproteininteractionhasbeenobservedinE.
coli,Streptomycescoelicolor,andMycobacteriumtuberculosis[21,51,52].
Interestingly,wediscoveredthatFtsINgonlyinteractswithFtsWNg,suggestingthatitslocalizationmaydependonthisprotein.
TheimportanceoftheuniqueFtsKNg-FtsNNginter-actioninN.
gonorrhoeae,asdeterminedbyB2H,isnotclear(Fig.
6,bluelines).
InE.
coli,FtsNisthelastpro-tein,oftenessentialcelldivisionproteins,recruitedtothedivisionsitetoinitiatecellconstriction[53,54].
ApreviousstudysuggestedthatE.
coliFtsNandFtsKstabilizetheZ-ringcooperatively,withoutdirectinterac-tions[55].
SincetheFtsK-FtsNinteractionispresentinN.
gonorrhoeae,theirjointinvolvementingonococcalcelldivisionrequiresfurtherinvestigation.
ZipANgdidnotinteractwithanyothergonococcalcelldivisionprotein.
InE.
coli,ZipAonlyinteractswithFtsZ,andisrequiredfordownstreamproteinrecruitment,in-cludingFtsK,FtsQ,FtsL,andFtsN[24,56].
OnereportsuggestedthatZipANgisahomologueoftheE.
coliproteinwithhighsimilarityinitskeydomains[57].
AlthoughZipANgcomplementedaconditionalzipAmutantinE.
coli,itdidnotfullyrestoreawildtypephenotypeinthisstrain[57].
Giventhesedata,theroleofZipAingonococcalcelldivisionremainstobeelucidated.
InN.
gonorrhoeae,theexistenceofFtsLNgisunclearduetoitslowhomologywithE.
coliFtsL[58].
Anopenreadingframe(ORF)locatedbetweenmraWandftsIinthedcwclusterofN.
gonorrhoeaewasreportedbyFrancisetal.
[7]andtheyreportedthatitwasnotacod-ingORF.
Snyderetal.
[58]namedthesameORFftsL.
BecausethisORFsharesonly17%aminoacidsimilaritytoitsE.
colihomologue,weconsideredthatitwasnotafunctionalORFanddidnottestitsinteractionwithothergonococcalcelldivisionproteins.
N.
gonorrhoeaelacksFtsB[7];thus,theproteincom-plexFtsQ-B-L,presentinotherspecies,suchasE.
coli,S.
pneumoniaeandB.
subtilis,wouldnotbeformedinN.
gonorrhoeae[59–61].
ThisproteincomplexhasbeendescribedasabridgeconnectingFtsKandtheFtsI-FtsWcomplexinE.
coli[18].
ArecentstudysuggeststhattheE.
coliFtsQ-B-Lcomplexactsasasignaltransmitterforcellwallremodelingandconstriction,whichismediatedbydirectinteractionswiththeFtsI-WcomplexandFtsN[19].
InS.
pneumoniae,theFtsQhomologue,DivIB,in-teractswithFtsKSp,FtsLSp,andFtsWSp[25].
Interest-ingly,ourB2HdatashowthatFtsQNgonlyinteractswithFtsANg,suggestingthatthefunctionofFtsQNgincelldivisioninN.
gonorrhoeaemaybedistinct.
Thereareseveralmodelsforbacterialcellconstriction.
OneE.
colimodelsuggeststhattheforcethatdrivesconstrictioncomesfromseptalpeptidoglycansynthesis[62].
Inthismodel,theFtsAEc-FtsNEcinteractionZouetal.
BMCMicrobiology(2017)17:232Page11of14activatespeptidoglycansynthesisbydirectorindirectinteractionwithFtsIEc[63].
AnotherE.
colimodelsug-geststhattheenergygeneratedfromFtsZ-mediatedGTPhydrolysisdrivescellconstriction[43].
Weob-servedanFtsANg-FtsNNginteractioninN.
gonorrhoeae.
However,thereisnofurtherevidencesupportingeithermodelofcellconstrictioninN.
gonorrhoeaeatthistime.
Thenon-essentialproteins,FtsENgandFtsXNg,arealsoimplicatedincelldivisioninN.
gonorrhoeae[64].
Similarly,inE.
coli,FtsEandFtsXarenon-essentialforcelldivisionunderconditionsofhighosmoticpressure[65].
GonococcalFtsEandFtsXhavehighsimilarityinaminoacidsequencetoknownhomologuesinotherspecies[64].
InE.
coli,theinteractionbetweenFtsEandFtsZhasaregulatoryeffectontheZ-ring[65].
FutureresearchcouldfocusonrevealingtheeffectsofFtsENgandFtsXNgoncelldivisioninN.
gonorrhoeae.
ThemajorissueinterpretingB2Hassayresultsistheempiricalcut-offof50%residual-galactosidaseactivityusedtodiscriminatepositiveandnegativeinteractions.
Inparticular,valuesclosetothecut-offcouldbeinter-pretedaseitherfalsepositiveornegativeresults.
Toval-idateourB2Hresults,weusedotherB2HinteractionstotestwhichsubdomainsofFtsANginteractedwithFtsZNg,FtsNNg,andFtsQNg.
Wedeterminedthatthe2Aand2BsubdomainsofFtsANginteractedwithFtsZNg,FtsQNg,andFtsNNg.
Wealsoevaluatedsomepositivein-teractionsobtainedbyB2HusingSPRandGSTpull-downassays.
TheSPRmethoddetectsandmeasuresweakortransientinteractions,inreal-time,withhighsensitivity[66].
TheSPRmethodshowedatransientFtsANg-FtsZNginteraction.
GSTpull-downassays,ontheotherhand,areidealindetectingstrongprotein-proteininteractions,asweakinteractionsmaydissociateduringtheassay[67].
Weconsiderthistobeareason-ableexplanationforourfailuretoconfirmwhentheinteractionofFtsANgwithFtsZNgwhenusingaGSTpull-downassay.
Todate,mostofstudiesoncelldivisionhavebeenfocusedonmodelorganisms(i.
e.
theGram-negativerodE.
coliandtheGram-positiverodB.
subtilis)duetotheabundantavailabilityoftoolsforgeneticma-nipulation[62].
Researchoncelldivisioninnon-modelorganismsisexpanding,andthisincludesstud-ieswithN.
gonorrhoeae[7,27].
Forexample,Chla-mydiatrachomatis,whichlacksFtsZ,requiresanactin-likeprotein,MreB,forcelldivision[68].
Ageneclusterencodingthreecelldivisionproteins,namedMldA,MldB,andMldC,wasidentifiedonlyinClostridiumdifficileanditscloselyrelatedbacteria[69].
Resultsfromstudiesusingnon-modelorganismssuggestthatcelldivisionmechanismsarecomplexandvaryindifferentorganisms,reflectingvastbio-logicaldiversity.
ConclusionsInourresearch,wediscoveredthatnineinteractionsamongeightcelldivisionproteinsdefinedthecelldiv-isioninteractomeofN.
gonorrhoeae.
IncomparisonwiththepublishedcelldivisioninteractomesofE.
coliandS.
pneumoniae,FtsA-FtsZandFtsZ-FtsKinteractionswerecommontoallthreebacteria.
FtsK-FtsNandFtsA-FtsWinteractionswereonlypresentinN.
gonorrhoeae,sug-gestingthattheyplaydifferentrolesinthecelldivisionofthismicroorganism.
ZipANgdidnotinteractwithanyothercelldivisionproteinstestedinthisstudy,indicat-ingthatitsrolemaydifferascomparedtoitsE.
colihomologue.
WealsodeterminedthatthesubdomainsofFtsANgwhichinteractedwithFtsQNg,FtsZNg,FtsWNg,orFtsNNg,differedfromitsE.
colihomologue.
ThissuggeststhatN.
gonorrhoeaepossessesadistinctivecelldivisioninteractome,andlikelyadifferentmechanismofcelldivisionascomparedtoE.
coliandotherorganisms.
AdditionalfilesAdditionalfile1:FigureS1.
SchematicrepresentationofN.
gonorrhoeaeftsAanditstruncations[33].
T1(162aa,Met1-Ala162)containedtheN-terminal1Aand1CdomainsofftsANg.
T2(194aa,Met1-Val194)includedtheN-terminal1A,1Cand1AdomainsofftsANg.
T3(230aa,Met1-Ile230)includedtheN-terminal1A,1C,1Aand2A1domainsofftsANg.
T4(71aa,Pro231-Glu301)containedthe2BdomainofftsANg.
T5(114aa,Ile301-Leu414)containedthe2A2and1AC-terminaldomainsofftsANg.
T6(64aa,Ala351-Leu414)containedthe1AC-terminaldomainofftsANg.
(DOCX30kb)Additionalfile2:FigureS2.
FtsZNgpolymerizationassays.
FtsZNgpolymersvisualizedbytransmissionelectronmicroscopewith(A)orwithout(B)2mMGTPinMESbuffer(50mMMES-NaOH,50mMKCl,10mMMgCl2,pH7.
5)at30°C.
SolidarrowsindicateFtsZNgpolymers.
Scalebarindicates100nm.
(C)LightscatteringofFtsZNgpolymerization(6μM)inMESbuffer.
(DOCX211kb)Additionalfile3:FigureS3.
InteractionsbetweenFtsANgtruncations(T2,T3,T4,T5andT6)andFtsWNg(W)byB2Hassay.
Valuesoflessthan50%(1250MillerUnits)indicateanegativeinteraction(greenbars).
(DOCX66kb)AbbreviationsB2H:Bacterialtwo-hybrid;Bs:Bacillussubtilis;Cs:Caulobactercrescentus;dcw:divisionandcellwall;DLS:Dynamiclightscattering;Ec:Escherichiacoli;Ng:Neisseriagonorrhoeae;ORF:Openreadingframe;Sp:Streptococcuspneumoniae;SPR:Surfaceplasmonresonance;TEM:Transmissionelectronmicroscopy;Y2H:Yeasttwo-hybridAcknowledgementsTheauthorswishtogivespecialthankstoJasonMaleyfromtheSaskatchewanStructuralSciencesCentre,UniversityofSaskatchewanforhelpwithsurfaceplasmonresonanceanddynamiclightscatteringexperiments.
ElectronmicroscopywasperformedattheWesternCollegeofVeterinaryMedicine,UniversityofSaskatchewan.
FundingThisworkwassupportedbytheSaskatchewanHealthResearchFoundationNewInvestigatorEstablishmentGrant(Grant#1866–2007toJRD)aswellastheNaturalSciencesandEngineeringResearchCouncilofCanadaGrant(Grant#203651–2012RGPINtoJRD).
YanLiandYinanZouwerepartiallysupportedbygraduatescholarshipsfromtheUniversityofSaskatchewan.
Zouetal.
BMCMicrobiology(2017)17:232Page12of14AvailabilityofdataandmaterialsThedatafromthisreportareincludedwithinthearticle.
Datasetsusedinthecurrentstudyareavailableuponrequest.
Authors'contributionsYZandYLparticipatedintheexperimentaldesign,implementationanddataanalysis.
YZalsowrotethefirstdraftofmanuscript.
JRDdesignedandsupervisedtheentireprojectandwasresponsibleforthefinalsubmissionofthemanuscript.
Allauthorscontributedtomanuscriptrevisions.
Allauthorshavereadandapprovedthefinalmanuscript.
EthicsapprovalandconsenttoparticipateEthicsapprovalisnotrequiredforthisstudy.
Consenttoparticipateisnotapplicable.
ConsentforpublicationNotapplicable.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Publisher'sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
Authordetails1DepartmentofMicrobiologyandImmunology,CollegeofMedicine,Saskatoon,SKS7N5E5,Canada.
2VaccineandInfectiousDiseaseOrganization,InternationalVaccineCentre,Saskatoon,SKS7N5E3,Canada.
3DepartmentofBiology,CollegeofArtsandScience,UniversityofSaskatchewan,Saskatoon,SKS7N5A5,Canada.
Received:12June2017Accepted:1December2017References1.
LutkenhausJ,AddinallSG.
BacterialcelldivisionandtheZring.
AnnuRevBiochem.
1997;66:93–116.
2.
MargolinW.
Themesandvariationsinprokaryoticcelldivision.
FEMSMicrobiolRev.
2000;24:531–48.
3.
AyalaJ,GarridoT,depedroMA,VicenteM.
Molecularbiologyofbacterialseptation.
In:GhuysenJM,HakenbeckR,editors.
NewcomprehensiveBiochemistry;1994.
p.
73–101.
4.
MingoranceJ,TamamesJ,VicenteM.
Genomicchannelinginbacterialcelldivision.
JMolRecognit.
2004;17:481–7.
5.
RealG,HenriquesAO.
LocalizationoftheBacillussubtilismurBgenewithinthedcwclusterisimportantforgrowthandsporulation.
JBacteriol.
2006;188:1721–32.
6.
MassiddaO,AnderluzziD,FriedliL,FegerG.
UnconventionalorganizationofthedivisionandcellwallgeneclusterofStreptococcuspneumoniae.
Microbiology.
1998;144:3069–78.
7.
FrancisF,Ramirez-ArcosS,SalimniaH,VictorC,DillonJA.
Organizationandtranscriptionofthedivisioncellwall(dcw)clusterinNeisseriagonorrhoeae.
Gene.
2000;251:141–51.
8.
TamamesJ,González-MorenoM,MingoranceJ,ValenciaA,VicenteM.
Bringinggeneorderintobacterialshape.
TrendsGenet.
2001;17:124–6.
9.
LutkenhausJ,PichoffS,DuS.
Bacterialcytokinesis:fromZringtodivisome.
Cytoskeleton(Hoboken).
2012;69:778–90.
10.
HaeusserDP,MargolinW.
Splitsville:structuralandfunctionalinsightsintothedynamicbacterialZring.
NatRevMicrobiol.
2016;14:305–19.
11.
ErringtonJ,DanielRA,ScheffersDJ.
Cytokinesisinbacteria.
MicrobiolMolBiolRev.
2003;67:52–65.
12.
GrengaL,RizzoA,PaolozziL,GhelardiniP.
Essentialandnon-essentialinteractionsininteractomenetworks:theEscherichiacolidivisionproteinsFtsQ-FtsNinteraction.
EnvironMicrobiol.
2013;15:3210–7.
13.
MukherjeeM,LutkenhausJ.
DynamicassemblyofFtsZregulatedbyGTPhydrolysis.
EMBOJ.
1998;17:462–9.
14.
HaleCA,deBoerPA.
DirectbindingofFtsZtoZipA,anessentialcomponentoftheseptalringstructurethatmediatescelldivisioninE.
coli.
Cell.
1997;88:175–85.
15.
GoehringNW,GonzalezMD,BeckwithJ.
Prematuretargetingofcelldivisionproteinstomidcellrevealshierarchiesofproteininteractionsinvolvedindivisomeassembly.
MolMicrobiol.
2006;61:33–45.
16.
BeggKJ,DewarSJ,DonachieWD.
AnewEscherichiacolicelldivisiongene,ftsK.
JBacteriol.
1995;177:6211–22.
17.
XCY,WeiheEK,MargolinW.
RoleoftheCterminusofFtsKinEscherichiacolichromosomesegregation.
JBacteriol.
1998;180:6424–8.
18.
BuddelmeijerN,BeckwithJ.
AcomplexoftheEscherichiacolicelldivisionproteinsFtsL,FtsBandFtsQformsindependentlyofitslocalizationtotheseptalregion.
MolMicrobiol.
2004;52:1315–27.
19.
TsangMJ,BernhardtTG.
ArolefortheFtsQLBcomplexincytokineticringactivationrevealedbyanftsLallelethatacceleratesdivision.
MolMicrobiol.
2015;95:925–44.
20.
MohammadiT,vanDamV,SijbrandiR,VernetT,ZapunA,BouhssA,etal.
IdentificationofFtsWasatransporteroflipid-linkedcellwallprecursorsacrossthemembrane.
EMBOJ.
2011;30:1425–32.
21.
FraipontC,AlexeevaS,WolfB,vanderPloegR,SchloesserM,denBlaauwenT,etal.
TheintegralmembraneFtsWproteinandpeptidoglycansynthasePBP3formasubcomplexinEscherichiacoli.
Microbiology.
2011;157:251–9.
22.
VicenteM,RicoAI.
Theorderofthering:assemblyofEscherichiacolicelldivisioncomponents.
MolMicrobiol.
2006;61:5–8.
23.
KarimovaG,DautinN,LadantD.
InteractionnetworkamongEscherichiacolimembraneproteinsinvolvedincelldivisionasrevealedbybacterialtwo-hybridanalysis.
JBacteriol.
2005;187:2233–43.
24.
DiLalloG,FagioliM,BarionoviD,GhelardiniP,PaolozziL.
Useofatwo-hybridassaytostudytheassemblyofacomplexmulticomponentproteinmachinerybacterialseptosomedifferentiation.
Microbiology.
2003;149:3353–9.
25.
MaggiS,MassiddaO,LuziG,FaddaD,PaolozziL,GhelardiniP.
Divisionproteininteractionweb:identificationofaphylogeneticallyconservedcommoninteractomebetweenStreptococcuspneumoniaeandEscherichiacoli.
Microbiology.
2008;154:3042–52.
26.
TapsallJW,NdowaF,LewisDA,UnemoM.
Meetingthepublichealthchallengeofmultidrugandextensivelydrug-resistantNeisseriagonorrhoeaethatcausesgonorrheainhumans.
ExpertRevAnti-InfectTher.
2009;7:821–34.
27.
SalimniaH,RadiaA,BernatchezS,BeveridgeTJ,DillonJA.
CharacterizationoftheftsZcelldivisiongeneofNeisseriagonorrhoeae:expressioninEscherichiaColiandN.
gonorrhoeae.
ArchMicrobiol.
2000;173:10–20.
28.
Ramirez-ArcosS,SzetoJ,BeveridgeTJ,VictorC,FrancisF,DillonJA.
Deletionofthecell-divisioninhibitorMinCresultsinlysisofNeisseriagonorrhoeae.
Microbiology.
2001;147:225–37.
29.
SzetoJ,Ramirez-ArcosS,RaymondC,HicksLD,KayCM,DillonJA.
GonococcalMinDaffectscelldivisioninNeisseriagonorrhoeaeandEscherichiacoliandexhibitsanovelself-interaction.
JBacteriol.
2001;183:6253–64.
30.
DiLalloG,CastagnoliL,GhelardiniP,PaolozziL.
Atwo-hybridsystembasedonchimericoperatorrecognitionforstudyingproteinhomoheterodimerizationinEscherichiacoli.
Microbiol.
2001;147:1651–6.
31.
KelloggDS,PeacockWL,DeaconWE,BrownL,PirkleDI.
Neisseriagonorrhoeae.
I.
Virulencegeneticallylinkedtoclonalvariation.
JBacteriol.
1963;85:1274–9.
32.
Greco-StewartV,Ramirez-ArcosS,LiaoM,DillonJR.
NterminusdeterminantsofMinCfromNeisseriagonorrhoeaemediateinteractionwithFtsZbutdonotaffectinteractionwithMinDorhomodimerization.
ArchMicrobiol.
2007;187:451–8.
33.
ZouY,LiY,EkanayakeSB,DillonJR.
AnEscherichiacoliexpressionmodelrevealsthespecies-specificfunctionofFtsAfromNeisseriagonorrhoeaeincelldivision.
FEMSMicrobiolLett.
2017;364:fnx078.
34.
KrolE,ScheffersDJ.
FtsZpolymerizationassays:simpleprotocolsandconsiderations.
JVisExp.
2013;81:e50844.
35.
LooseM,MitchisonTJ.
ThebacterialcelldivisionproteinsFtsAandFtsZself-organizeintodynamiccytoskeletalpatterns.
NatCellBiol.
2014;16:38–46.
36.
PichoffS,LutkenhausJ.
TetheringtheZringtothemembranethroughaconservedmembranetargetingsequenceinFtsA.
MolMicrobiol.
2005;55:1722–34.
37.
BeuriaTK,MullapudiS,MileykovskayaE,SadasivamM,DowhanW,MargolinW.
Adeninenucleotide-dependentregulationofassemblyofbacterialtubulin-likeFtsZbyahypermorphofbacterialactin-likeFtsA.
JBiolChem.
2009;284:14079–86.
38.
JensenSO,ThompsonLS,HarryEJ.
CelldivisioninBacillussubtilis:FtsZandFtsAassociationisZ-ringindependent,andFtsAisrequiredforefficientmidcellZ-ringassembly.
JBacteriol.
2005;187:6536–44.
Zouetal.
BMCMicrobiology(2017)17:232Page13of1439.
YanK,PearceKH,PayneDJ.
AconservedresidueattheextremeC-terminusofFtsZiscriticalfortheFtsA-FtsZinteractioninStaphylococcusaureus.
BiochemBiophysResCommun.
2000;270:387–92.
40.
GambaP,VeeningJW,SaundersNJ,HamoenLW,DanielRA.
Two-stepassemblydynamicsoftheBacillussubtilisdivisome.
JBacteriol.
2009;191:4186–94.
41.
DinN,QuardokusEM,SackettMJ,BrunYV.
DominantC-terminaldeletionsofFtsZthataffectitsabilitytolocalizeinCaulobacteranditsinteractionwithFtsA.
MolMicrobiol.
1998;27:1051–63.
42.
RuedaS,VicenteM,MingoranceJ.
ConcentrationandassemblyofthedivisionringproteinsFtsZ,FtsA,andZipAduringtheEscherichiacolicellcycle.
JBacteriol.
2003;185:3344–51.
43.
OsawaM,AndersonDE,EricksonHP.
CurvedFtsZprotofilamentsgeneratebendingforcesonliposomemembranes.
EMBOJ.
2009;28:3476–84.
44.
ArumugamS,ChwastekG,Fischer-FriedrichE,EhrigC,MnchI,SchwilleP.
SurfacetopologyengineeringofmembranesforthemechanicalinvestigationofthetubulinhomologueFtsZ.
AngewChemIntEdEng.
2012;51:11858–62.
45.
WangSC,WestL,ShapiroL.
ThebifunctionalFtsKproteinmediateschromosomepartitioningandcelldivisioninCaulobacter.
JBacteriol.
2006;188:1497–508.
46.
BillerSJ,BurkholderWF.
TheBacillussubtilisSftA(YtpS)andSpoIIIEDNAtranslocasesplaydistinctrolesingrowingcellstoensurefaithfulchromosomepartitioning.
MolMicrobiol.
2009;74:790–809.
47.
SherrattDJ,ArciszewskaLK,CrozatE,GrahamJE,GraingeI.
TheEscherichiacoliDNAtranslocaseFtsK.
BiochemSocTrans.
2010;38:395–8.
48.
GrengaL,LuziG,PaolozziL,GhelardiniP.
TheEscherichiacoliFtsKfunctionaldomainsinvolvedinitsinteractionwithitsdivisomeproteinpartners.
FEMSMicrobiolLett.
2008;287:163–7.
49.
MercerKL,WeissDS.
TheEscherichiacolicelldivisionproteinFtsWisrequiredtorecruititscognatetranspeptidase,FtsI(PBP3),tothedivisionsite.
JBacteriol.
2002;184:904–12.
50.
MohammadiT,SijbrandiR,LuttersM,VerheulJ,MartinNI,denBlaauwenT,etal.
SpecificityofthetransportoflipidIIbyFtsWinEscherichiacoli.
JBiolChem.
2014;289:14707–18.
51.
MistryBV,DelSolR,WrightC,FindlayK,DysonP.
FtsWisadispensablecelldivisionproteinrequiredforZ-ringstabilizationduringsporulationseptationinStreptomycescoelicolor.
JBacteriol.
2008;190:5555–66.
52.
DattaP,DasguptaA,SinghAK,MukherjeeP,KunduM,BasuJ.
InteractionbetweenFtsWandpenicillin-bindingprotein3(PBP3)directsPBP3tomid-cell,controlscellseptationandmediatestheformationofatrimericcomplexinvolvingFtsZ,FtsWandPBP3inmycobacteria.
MolMicrobiol.
2006;62:1655–73.
53.
GerdingMA,LiuB,BendezuFO,HaleCA,BernhardtTG,deBoerPA.
Self-enhancedaccumulationofFtsNatdivisionsitesandrolesforotherproteinswithaSPORdomain(DamX,DedD,andRlpA)inEscherichiacolicellconstriction.
JBacteriol.
2009;191:7383–401.
54.
RicoAI,Garcia-OvalleM,PalaciosP,CasanovaM,VicenteM.
RoleofEscherichiacoliFtsNproteinintheassemblyandstabilityofthecelldivisionring.
MolMicrobiol.
2010;76:760–71.
55.
GoehringNW,RobichonC,BeckwithJ.
RoleforthenonessentialNterminusofFtsNindivisomeassembly.
JBacteriol.
2007;189:646–9.
56.
HaleCA,deBoerPAJ.
ZipAisrequiredforrecruitmentofFtsK,FtsQ,FtsL,andFtsNtotheseptalringinEscherichiacoli.
JBacteriol.
2002;184:2552–6.
57.
DuY,ArvidsonCG.
IdentificationofZipA,asignalrecognitionparticle-dependentproteinfromNeisseriagonorrhoeae.
JBacteriol.
2003;185:2122–30.
58.
SnyderLA,SaundersNJ,ShaferWM.
Aputativelyphasevariablegene(dca)requiredfornaturalcompetenceinNeisseriagonorrhoeaebutnotNeisseriameningitidisislocatedwithinthedivisioncellwall(dcw)genecluster.
JBacteriol.
2001;183:1233–41.
59.
DanielRA,Noirot-GrosMF,NoirotP,ErringtonJ.
MultipleinteractionsbetweenthetransmembranedivisionproteinsofBacillussubtilisandtheroleofFtsLinstabilityindivisomeassembly.
JBacteriol.
2006;188:7396–404.
60.
Noirclerc-SavoyeM,LeGouellecA,MorlotC,DidebergO,VernetT,ZapunA.
InVitroreconstitutionofatrimericcomplexofDivIB,DivICandFtsL,andtheirtransientco-localizationatthedivisionsiteinStreptococcuspneumoniae.
MolMicrobiol.
2005;55:413–24.
61.
RobichonC,KingGF,GoehringNW,BeckwithJ.
ArtificialseptaltargetingofBacillussubtiliscelldivisionproteinsinEscherichiacoli:aninterspeciesapproachtothestudyofprotein-proteininteractionsinmultiproteincomplexes.
JBacteriol.
2008;190:6048–59.
62.
EswaraPJ,RamamurthiKS.
Bacterialcelldivision:nonmodelspoisedtotakethespotlight.
AnnuRevMicrobiol.
2017;71:393–411.
63.
LiuB,PersonsL,LeeL,deBoerPA.
RolesforbothFtsAandtheFtsBLQsubcomplexinFtsN-stimulatedcellconstrictioninEscherichiacoli.
MolMicrobiol.
2015;95:945–70.
64.
BernatchezS,FrancisF,SalimniaH,BeveridgeTJ,LiH,DillonJA.
Genomic,transcriptionalandphenotypicanalysisofftsEandftsXofNeisseriagonorrhoeae.
DNARes.
2000;7:75–81.
65.
CorbinBD,WangY,BeuriaTK,MargolinW.
InteractionbetweencelldivisionproteinsFtsEandFtsZ.
JBacteriol.
2007;189:3026–35.
66.
NgounouWetieAG,SokolowskaI,WoodsAG,RoyU,LooJA,DarieCC.
Investigationofstableandtransientprotein-proteininteractions:past,present,andfuture.
Proteomics.
2013;13:538–57.
67.
BrucknerA,PolgeC,LentzeN,AuerbachD,SchlattnerU.
Yeasttwo-hybrid,apowerfultoolforsystemsbiology.
IntJMolSci.
2009;10:2763–88.
68.
LiechtiG,KuruE,PackiamM,HsuYP,TekkamS,HallE,etal.
Pathogenicchlamydialackaclassicalsacculusbutsynthesizeanarrow,mid-cellpeptidoglycanring,regulatedbyMreB,forcelldivision.
PLoSPathog.
2016;12:e1005590.
69.
RansomEM,WilliamsSB,WeissDS,EllermeierCD.
Identificationandcharacterizationofageneclusterrequiredforproperrodshape,celldivision,andpathogenesisinClostridiumdifficile.
JBacteriol.
2014;196:2290–300.
70.
MirouxB,WalkerJE.
Over-productionofproteinsinEscherichiacoli:mutanthoststhatallowsynthesisofsomemembraneproteinsandglobularproteinsathighlevels.
JMolBiol.
1996;260:289–98.
71.
PicardFJ,DillonJA.
BiochemicalandgeneticstudieswitharginineandprolineauxotypesofNeisseriagonorrhoeae.
CanJMicrobiol.
1989;35:1069–75.
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com/submitSubmityournextmanuscripttoBioMedCentralandwewillhelpyouateverystep:Zouetal.
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