Kois-Ostrowska-etal-PLOSG2016-Bacterial-chromosome-segregation-machinery-in-apically

44 Pages·2017·5.76 MB·English

RESEARCHARTICLE
Unique Function of the Bacterial
Chromosome Segregation Machinery in
Apically Growing Streptomyces - Targeting the
Chromosome to New Hyphal Tubes and its
Anchorage at the Tips
AgnieszkaKois-Ostrowska1☯,AgnieszkaStrzałka1☯,NataliaLipietta1,EmmaTilley2,
JolantaZakrzewska-Czerwińska1,3,PaulHerron2,DagmaraJakimowicz1,3*
a11111
1 FacultyofBiotechnology,UniversityofWroclaw,Poland,2 InstituteofPharmacyandBiomedicalSciences,
UniversityofStrathclyde,Glasgow,UnitedKingdom,3 InstituteofImmunologyandExperimentalTherapy,
Wroclaw,Poland
☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS
Abstract
Citation:Kois-OstrowskaA,StrzałkaA,LipiettaN,
TilleyE,Zakrzewska-CzerwińskaJ,HerronP,etal.
(2016)UniqueFunctionoftheBacterial Thecoordinationofchromosomesegregationwithcellgrowthisfundamentaltotheprolifer-
ChromosomeSegregationMachineryinApically ationofanyorganism.Inmostunicellularbacteria,chromosomesegregationisstrictlycoor-
GrowingStreptomyces-Targetingthe
dinatedwithcelldivisionandinvolvesParAthatmovestheParBnucleoproteincomplexes
ChromosometoNewHyphalTubesandits
bi-orunidirectionallytowardthecellpole(s).However,thechromosomeorganizationinmul-
AnchorageattheTips.PLoSGenet12(12):
e1006488.doi:10.1371/journal.pgen.1006488 tiploid,apicallyextendingandbranchingStreptomyceshyphaechallengestheknownmech-
anismsofbacterialchromosomesegregation.ThecomplexStreptomyceslifecycle
Editor:PatrickH.Viollier,UniversityofGeneva
MedicalSchool,SWITZERLAND involvestwostages:vegetativegrowthandsporulation.Inthelatterstage,multiplecelldivi-
sionsaccompaniedbychromosomecompactionandParABassistedsegregationturnmulti-
Received:September8,2016
genomichyphalcellintoachainofunigenomicspores.However,therequirementforactive
Accepted:November16,2016
chromosomesegregationisunclearintheabsenceofcanonicalcelldivisionduringvegeta-
Published:December15,2016
tivegrowthexceptintheprocessofbranchformation.Themechanismbywhichchromo-
Copyright:©2016Kois-Ostrowskaetal.Thisisan somesaretargetedtonewhyphaeinstreptomycetevegetativegrowthhasremained
openaccessarticledistributedunderthetermsof
unknownuntilnow.Here,weaddressthequestionofwhetheractivechromosomesegrega-
theCreativeCommonsAttributionLicense,which
tionoccursatthisstage.AppliedforthefirsttimeinStreptomyces,labellingofthechromo-
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal somalreplicationinitiationregion(oriC)andtime-lapsemicroscopy,revealedthatin
authorandsourcearecredited. vegetativehyphaeeverycopyofthechromosomeiscomplexedwithParB,whereasParA,
DataAvailabilityStatement:Allrelevantdataare throughinteractionwiththeapicalproteincomplex(polarisome),tightlyanchorsonlyone
withinthepaperanditsSupportingInformation chromosomeatthehyphaltip.Theanchorismaintainedduringreplication,whenParAcap-
files.
turesoneofthedaughteroriCs.Duringsporegerminationandbranching,ParAtargetsone
Funding:ThisworkwasfundedbyMaestro ofthemultiplechromosomalcopiestothenewhyphaltip,enablingefficientelongationof
fundingscheme,grant2012/04/A/NZ1/00057from
hyphaltube.Thus,ourstudiesrevealanovelroleforParABproteinsduringhyphaltipestab-
NationalScienceCentreawardedtoJZC,Poland,
co-financedbyHarmoniagrant2014/14/M/NZ1/ lishmentandextension.
00076fromNationalScienceCentre,Poland
awardedtoDJ.EJTwasfinancedbyBBSRC(UK)
grantBB/D521657/1awardedtoPRH.Thecostof
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 1/25
ChromosomeSegregationduringHyphalGrowth
publicationwasfinancedbytheWroclawCentreof
Biotechnology,programmeoftheLeadingNational AuthorSummary
ResearchCentre(KNOW)foryears2014–2018.
Toproliferate,cellssynchronizegrowthanddivisionwithchromosomesegregation.In
Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or unicellularbacteria,chromosomessegregateduringreplicationbyactivemovementof
preparationofthemanuscript. nucleoproteincomplexestowardthecellpole(s).Here,weaskedthequestionhowactive
chromosomesegregationoccursintheabsenceofcelldivision,duringhyphalgrowthand
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. branchingofthefilamentousbacterium,Streptomycescoelicolor.Weshowthatinmultige-
nomicStreptomyceshyphae,thebacterialsegregationmachineryanchorsasinglechromo-
someatthehyphaltip.Throughchromosomalanchorage,segregationproteinsfacilitate
chromosometargetingtothenewlyformedgermtubesorbranches.Thus,beingadapted
forapicalgrowth,inStreptomyceshyphaethebacterialsegregationmachineryimposesa
chromosomedistributionthatisreminiscentofnucleardistributioninapicallygrowing
eukaryoticcellssuchasfilamentousfungi.
Introduction
Chromosomesegregationinunicellularbacteriaisstrictlycoordinatedwiththecellcycleand
chromosomesaresegregatedduringtheirreplicationandpriortocelldivision.However,the
spatialchromosomeorganizationinbacteria,determinedbythepositionoftheoriginofrepli-
cation(oriC)inthecell,differswithrespecttothemorphologyandgrowthstrategyofthe
organism[1–3].Thesedifferencesinchromosomeorganizationarereflectedinthespecific,
cellcycle-tunedmechanismofchromosomesegregation[4,5].Inmostbacterialspecies(with
theexceptionofEscherichiacoliandsomeγ-proteobacteria)efficientchromosomesegregation
reliesontheactivityoftwoproteins,ParAandParB[4,6].BybindingparSsitesclustered
aroundoriC,ParBassemblesthisregionofthechromosomeintoalargenucleoproteincom-
plex.Soonaftertheinitiationofreplication,ParBcomplexesaresegregatedintospecificloca-
tionsofthecellduetointeractionwiththeATPase,ParA[6–8].Species-specificdifferencesin
spatialchromosomeorganizationarelinkedwithvariationsintheParAandParBchoreogra-
phy.Forinstance,duringvegetativegrowthofBacillussubtilis,ParBcomplexesaresegregated
bi-directionallytooppositecellpoles,whileinCaulobactercrescentusandinthecaseofVibrio
choleraechromosomeI,onlyoneofthetwoParB/oriCnucleoproteincomplexesismoved
towardtheoppositepolebytheParAassembly[2].TheinteractionofParAwithproteins
localizedatthecellpole,suchasPopZandTipNinC.crescentusorHubPinV.cholerae,trans-
latethecellpolaritytoasymmetricchromosomesegregation[9–11].Thechromosome
arrangementandmechanismofsegregationremainsunexploredinmultigenomicbacteria
suchasthefilamentousactinobacteriaincludingStreptomyces.
Streptomycesdifferremarkablyfromotherbacteriawiththeirhyphalgrowththatisreminis-
centoffilamentousfungi[12].Moreover,Streptomyceselongatedhyphalcellscontainmultiple
copiesoflinearchromosomes.Duringcolonydevelopment,twotypesofhyphalcellsarepro-
duced:branchingvegetativehyphaethatformadensemycelialnetworkandsporogenic
hyphae,thatareconvertedintochainsofsporesbymultiplecelldivisions.Incontrasttomost
bacteriathatextendalongthelateralcellwall,Streptomycesasotheractinobacteria,growby
cellextensionatthepoles(tips)[13,14].TheapicalsynthesisofpeptidoglycaninActinobac-
teriaislinkedtotheactivityoftheessentialcoiled-coilprotein,DivIVAthatlocalizesatthecell
poles[15–17].WhatisuniquetoStreptomycesgrowth,istheunidirectionalcellextensionat
thehyphaltips.InStreptomyces,polargrowthisdirectedbyaproteincomplexlocalizedatthe
hyphaltip(‘polarisome’ortip-organizingcomplex,TIPOC),whichincludesDivIVAand
anothercoiled-coilprotein,Scy[16–18].Branchingisinitiatedbyassemblyofthepolarisome
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 2/25
ChromosomeSegregationduringHyphalGrowth
atasiteonthelateralwalldistantfromtheextendingtip[14,19].Asimilarmechanismof
branchformationwasobservedinthefilamentousfungusNeurospora crassa,suggestingthatit
isafeaturesharedbetweenbacteriaandeukaryote[20].
ThetwostagesofStreptomycesdevelopment,vegetativegrowthandsporulation,differwith
respecttothecellularorganizationandcellcycleevents.Duringsporulation,multiple,syn-
chronizeddivisionsofelongatedsporogeniccellsareaccompaniedbycondensationandsegre-
gationofnumerouschromosomalcopies[12].Ourearlierstudiesshowedthatthesegregation
proteinsParAandParBuniformlydistributechromosomesalongthelongsporogeniccellat
thetimeofitsseptation[21,22].Duringvegetativegrowth,multigenomichyphalcells,named
hyphalcompartments,donotundergotypicalcelldivision.Widelyspacedcrosswallsdelimit,
butdonotseparate,hyphalcompartmentswhichremainadjacentinlonghyphae[23,24].
Verylittleisknownaboutchromosomeorganizationinvegetativehyphae.Severalcopiesof
thechromosomesremainuncondensedandvisiblyunseparatedwhenvisualizedbyDNA
staininginhyphalcompartments.FISH(fluorescenceinsituhybridization)experimentsindi-
catedthattheendsoflinearchromosomesarespatiallycloseandthechromosomesare
unevenlydistributedinvegetativehyphae[25].Inaddition,replisomelabelingdemonstrated
thatchromosomesreplicateasynchronouslywithinthecompartmentsandfollowtheextend-
ingtip[27,28].Localizationofsegregationproteinsinvegetativehyphaeissignificantlydiffer-
entfromtheirlocalizationinsporulatinghyphae.ParBwasvisualizedasmultiple,irregularly
spacedcomplexes,withadistinctfocuslocatedataconstantdistancefromthehyphaltip[26].
Meanwhile,ParAinvegetativehyphae,localizesexclusivelyatthehyphaltips(notalongthe
cellasinsporogeniccells),whereitinteractswithScy[29].Eventhoughthelocalizationofthe
segregationproteinssuggestedtheirengagementintheorganizationoftheapicalchromo-
some,chromosomedistributionandtheroleofParAandParBduringgrowthofmultige-
nomicvegetativehyphaeremainsunknownintheabsenceofchromosomallocus-specific
labellingtools.
Tounderstandhowhyphaltipextensionandbranchingarecoordinatedwithdistribution
andsegregationofmultiplechromosomalcopiesduringvegetativegrowthofS.coelicolor,we
tookadvantageoforiClabelingandtime-lapsefluorescentmicroscopy.Weshowthatinmulti-
genomichyphalcellsParAanchorsthesingleapicalchromosomeandunidirectionallysegre-
gatesoneofthenewlyreplicatedoriCregionsatthetip.Duringestablishmentofthenew
hyphaltip,ParA-mediatedapicaloriCanchoragetargetsachromosomefrommultigenomic
celltoanewbranchoragermtube.Ourstudyrevealsauniquemechanismforbacterialchro-
mosomesegregationthatisadjustedtoaccommodatehyphalgrowthandbranching.
Results
AlloriCsinmultigenomicvegetativehyphaeareboundbyParB
ToaddressthequestionhowchromosomesaredistributedinS.coelicolorapicallyextending
andbranchingvegetativehyphaeweconstructedafluorescentreporter-operatorsystem
(FROS)tomarkchromosomaloriCregions.
AFROScassettethatcontainedanarrayof120tandemtetOrepeats[30]wasintegrated
intotheS.coelicolorchromosomeapproximately29kbfromtheoriCregionbyinvitrotrans-
positionandintergenicconjugation[31](Fig1A,S1AFig)resultinginthestrainEJTH31.Sub-
sequentlythetetR-mcherrygenewasintegratedintothechromosomeofthisstrainon
pMS83mCherryresultingintheFROSstrain,DJ-NL102(S1AFig,strainswereverifiedas
showninS1BandS1CFig).AnalysisofDJ-NL102hyphaerevealedirregularlydistributed
mCherryfoci(themeandistancebetweenfociwas2.0±1.2μm,S2AandS2BFig).Thefoci
disappeareduponadditionofanhydrotetracycline(aTc)totheculturemedium,presumably
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 3/25
ChromosomeSegregationduringHyphalGrowth
Fig1.ParB-EGFPcomplexesco-localizewithalloriCsinmultigenomicS.coelicolorvegetativehyphae.(A)SchemeofFROS
cassettelocalizationintheS.coelicolorchromosome.(B)ImagesofParB-EGFP(green)andFROS(red)fociinvegetativehyphaeofFROS
parB-egfpstrain(AK113).Thehyphaltipsaremarkedwithanasterisk,scalebar—1μm.(C)Co-localizationofFROSandParB-EGFPfociin
AK113strainalongthevegetativehyphaeandatthetipsofhyphae;thepercentageofthefocilocalizingwithinthegivendistanceis
indicated.(D)CorrelationbetweenthedistancefromParB-EGFPtothetipofhyphaeanddistancefromFROSsignaltotipofFROSparB-
egfp(AK113)strainhyphae.Thescatterplotwithafittedlinearmodelshowsdatafrom16hyphaemeasuredat10minutetimeintervals.
Datawereanalyzedusingamixedeffectsmodelwhichcancompensateforanindividualhyphaeffectandthestandardlinearmodel.
Resultsofbothmodelsweresimilar,comparisonofLog-likelihoodsofbothmodelsshowedthatrandomeffectsofindividualhyphaewere
notsignificant.CorrelationwascalculatedusingthePearsonmethod.
doi:10.1371/journal.pgen.1006488.g001
duetothereliefofTetRbindingtotetO(S2AFig)[30].However,incontrasttoE.coli[30],we
didnotdetectanygrowthimpairmentordisturbedreplicationofFROSstrain(s)inthe
absenceofaTc(S2C,S2D,S2EandS2FFig).Thissuggeststhat,atleastinvegetativehyphae,
thebindingofTetR-mCherrytothetetOarrayintheFROScassettedidnotcauseseriousrepli-
cationroadblocksdetrimentaltogrowth.
WeexpectedthatinhyphalcellsParB-EGFPshouldco-localizewithatleastsomeoriCsof
themultiplechromosomalcopies.Tocheckthis,weanalyzedastrainAK113withtheFROS
cassetteandtetR-mcherryexpressedinaparB-egfpbackground.InAK113hyphae,97%ofPar-
B-EGFPfociandmCherry-FROSfocioverlapped(distancebetweenfocilessthan1μm)(Fig
1Band1C).WedidnotobserveanyFROSfociunaccompaniedbytheParB-EGFPcomplexat
thehyphaltips,suggestingthatthetip-proximalchromosomeisconstantlyboundbyParB.
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 4/25
ChromosomeSegregationduringHyphalGrowth
Thus,theFROSlabelingoforiCregionsinS.coelicolorconfirmedirregulardistributionof
themultiplecopiesofchromosomesandindicatedbindingofParBtoeachchromosomaloriC
regioninthemultigenomichyphae.
OnlyapicaloriC-ParBcomplextightlyfollowstheextendinghyphaltip
EarlierstudiesofchromosomereplicationinS.coelicolorvegetativehyphaeshowedreplisome
traffickingandsuggestedthatchromosomesfollowtheextendingtip[27].Theconstantdis-
tancebetweentheFROS/ParBcomplexandthetip,observedinsnapshotanalysis,suggests
thatthetip-proximalchromosomeisanchoredtothetipduringhyphalextension.
Theapplicationoftime-lapsemicroscopyandtheFROSstrains(DJ-NL102andAK113)
allowedustoexaminethechromosomedistributionduringhyphalgrowth(Fig2,S3A,S3B,
S3CandS3DFig).Theearlierobservationsshowedthatreplicationstartsbeforesporegermi-
nation[27,28],andthegerminatingsporesshowedmultipleFROSfoci,asexpected.Inspore
germtubes(Fig2Atoppanel,S3AandS3DFigtoppanel,S1Movie)andextendingvegetative
hyphaeof24hoursoldcolonies(Fig2Abottompanel,S3DFig,bottompanel),thedistance
betweenthehyphaltipandthefirst,tip-proximalFROScomplexwasconstant(1.4±0.4μm),
demonstratingthatthefirstoriCfollowstheelongatingtip(Fig2B).Interestingly,thedistance
betweenthetipandtheFROScomplexeslocatedfurtherfromthetipwasmorevariableduring
hyphalextension(Fig2B,inset).Wecomparedthetip-proximal(oriC1)andtip-distalcom-
plexes(oriC2—oriC8)byanalyzingthecorrelationbetweentheirmovementandtipgrowth
rates(Fig2C).Thecorrelationwashighforthefirstchromosomeanddecreasedwithan
increasingdistancebetweentheoriCsandtheextendingtip.
Next,wemeasuredthedistancebetweenthehyphaltipandthefirstFROSsignalinextend-
inghyphaeofFROS,parB-egfpstrain(AK113)andplotteditagainstthedistancebetweenthe
ParBcomplexandthetip(Fig1D).ThisanalysisconfirmedthatthepositionsoftheParBand
FROSsignalsatthehyphaltipwerehighlycorrelatedandaveragedistancebetweenbothcom-
plexesandthetipwas1.5–2.0μm.
Thus,thetime-lapseanalysisconfirmedthatchromosomesfollowtheextendinghyphaltip.
Markedly,onlythefirstoriCremainstightlyassociatedwiththeextendinghyphaltipand
maintainsaconstantdistancetoit.
ParABproteinslocalizeoriCataconstantdistancetothetip
ParBbindstheoriCsofeachchromosomealongthehyphae,butonlythefirstonemaintainsa
constantdistanceandfollowstheextendinghyphaltip.AsParAinvegetativehyphaeislocal-
izedatthetip[21,29](S4Fig),wehypothesizedthatthepresenceofParAanditsinteraction
withthetip-proximalParBcomplexarecriticalforthelocalizationoftheoriC/ParBcomplex
attheconstantdistancefromthetip.AnalysisofFROS-markedoriCregionsintheΔparBand
ΔparAbackgroundallowedustoverifythis.
ThesnapshotsanalysisrevealedthatintheΔparBandΔparA(AK114andAK115)mutant
strains,thetip-proximalFROScomplexwasfurtherawayfromthehyphaltipthaninthewild
typeFROSstrain.Thedistancesbetweentip-proximalFROScomplexesandthetipwere
2.5±1.4μminΔparB,2.3±1.0μminΔparAand1.4±0.4μmin“wildtype”strain(Fig3A
and3B,differencesbetweenmutantsandthewildtypestrainwerestatisticallysignificant,
p<0.001,verifiedwithANOVAandaposthocGames-Howelltest).Moreover,thepositionof
theapicalFROScomplexexhibitedhighervariationinΔparBandΔparAstrainsthaninthe
wildtypestrain(demonstratedwithanF-test;theratioofvarianceswas6.3±1.2forΔparA
and11.9±2.3forΔparBinrelationtothewildtypestrain(p<2.2e-16)).Wealsoobservedthat
intheΔparAstrain,butsurprisinglynotintheΔparBstrain,thedistancebetweentheedgeof
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 5/25
ChromosomeSegregationduringHyphalGrowth
Fig2.Thetip-proximalchromosomefollowstheextendingvegetativehyphaetip.(A)Time-lapsesnapshotsoftheFROS
strain(DJ-NL102)germinatingspore(toppanel)andvegetativehypha(bottompanel).TheimagesaretheoverlayofTetR-mCherry
fluorescence(red)andDICimage(gray)(forseparateimagesofTetR-mCherryfluorescenceandDICseeS3DFig).Thearrows
indicate:red—oriC1(closesttothetipofthehypha),yellow—oriC2,purple—oriC3,asterisksindicatethetipofoutlinedhyphae,scale
bar—1μm.(B)PositionsoftheFROScomplexesintheextendinghyphaeofFROSstrain(DJ-NL102).Greybarsarerepresentations
oftheextendinghyphaewith95%confidenceintervalforhyphallengthandsemitransparentcoloreddotsrepresentoriCpositions
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 6/25
ChromosomeSegregationduringHyphalGrowth
(red–oriC1,yellow–oriC2,purple–oriC3,asshownintheschematicdrawingattheright),coloredlinesindicate95%mean
confidenceintervals(analyzedfor41hyphae).Inset:Distribution(shownasprobabilitydensityfunction)ofthedistancesbetweenthe
hyphaltipandtheoriC1(red),oriC2(yellow)andoriC3(purple).(C)CorrelationofhyphalextensionrateandFROScomplex
movementcalculatedfor8subsequentoriCsfromthetip.Scatterplotswithfittedlinearmodelsshowdatafrom20hyphaemeasured
at10minutetimeintervals,greyareaindicates95%confidenceintervalforthemodel.Minussignmeansthatthedistancebetween
thechromosomeandthetipisincreasingandaplussignthatitisdecreasing.Datawereanalyzedusingamixedeffectsmodel,
whichcancompensatefortheeffectofindividualhyphaeandastandardlinearmodel.Resultsofbothmodelswereverysimilar,
comparisonofLog-likelihoodsofbothmodelsshowedthatrandomeffectsofindividualhyphaewerenotsignificant.Inset:the
calculatedcorrelationinrelationtotheoriCpositioninthehyphaewithafittedlinearmodel.Correlationswerecalculatedusing
Pearsonmethodwith95%confidenceintervals.
doi:10.1371/journal.pgen.1006488.g002
nucleoidandthehyphaltipwasincreased,whencomparedtothewildtypeFROSstrain(S5
Fig,onlythedifferencebetweentheΔparAandthewildtypewasstatisticallysignificant,
p<0.001verifiedwithANOVAandposthocTukey’stest).
Theincreaseddistanceofthetip-proximaloriCtotheextendinghyphaltipintheΔparA
(AK115)andΔparB(AK114)strainswasconfirmedbytime-lapseanalysisofFROScomplex
dynamics(S6Fig).PlottingthecorrelationbetweenthehyphalextensionrateandoriCmove-
mentshowedthattheassociationofthefirstoriCwiththehyphaltipwasvisiblydecreasedin
themutantstrains(Fig3C).AlthoughchromosometraffickinginthehyphaeoftheΔparAand
ΔparBstrainswasmaintained,wenotedaslightlyincreasedvariationofthedistancesbetween
oriC2ororiC3andthetip(S6Fig).
Fig3.TheconstantdistancebetweenoriCandthehyphaltipisdependentonParAB.(A)ImagesofFROSinthe“wildtype”FROS
strain(DJ-NL102),ΔparAFROS(AK115)andΔparBFROS(AK114)strains.TheimagesaretheoverlayofTetR-mCherryfluorescence
(red)andDICimage(grey),asterisksindicatethetipofhyphae,scalebar—1μm.(B)Distribution(shownasprobabilitydensityfunction)of
thedistancesbetweenthehyphaltipandtip-proximalFROSsignalin“wildtype”FROS(DJ-NL102),ΔparAFROS(AK115)andΔparB
FROS(AK114)strains.(C)Correlationbetweenhyphalextensionrateandthetip-proximaloriCmovementvelocityin“wildtype”FROS
(DJ-NL102),ΔparAFROS(AK115)andΔparBFROS(AK114)strains(analyzedfor41ofDJ-NL102,31AK115and30AK114hyphae).
Scatterplotswithfittedlinearmodels,greyareaindicates95%confidenceintervalforthemodel.
doi:10.1371/journal.pgen.1006488.g003
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 7/25
ChromosomeSegregationduringHyphalGrowth
OuranalysesshowthatintheΔparAandΔparBmutantstrains,chromosometraffickingin
hyphaewasmaintainedalbeitslightlydisturbed.Moreover,bothsegregationproteinsParA
andParBwereessentialforanchorageofthetip-proximalchromosomeoriCregionatthetip
oftheextendinghyphae.
ParApositionsthetip-proximalParB–oriCcomplex
Havingestablishedthatbothsegregationproteinsarerequiredforthetip-proximaloriC
localization,wesoughtconfirmationthatParAanchorsandorganizesthefirstParBcom-
plex.Toaddressthisquestion,wecomparedthelocalizationoftheParBcomplexinasetof
strainswithdifferentparAmodifications:ΔparA(J3318),aparAoverexpressionstrain
(DJ532)andastrainwithamutationthatabolishestheinteractionwithParBandScy—
parA (DJ598)[29].
mut
InparAmutantstrains,thetip-proximalParBcomplexwasdelocalizedandpositionedfur-
therawayfromthetipthaninthewildtypestrain.ThedistancebetweentheParB-EGFPcom-
plexandthetipwas2.2±1.4μmintheΔparAstrain,3.0±1.5μminthestrainoverexpressing
parAand2.5±1.1μmintheparA strainincomparisonto1.7±0.8μminthecontrol
mut
parB–egfpstrain(Fig4Aand4D,thedifferencesbetweenthemutantsandthewildtypestrain
werestatisticallysignificant,p<0.001,calculatedwithANOVAandposthocGames-Howell
test).ThemeasureddistanceofParB-EGFPcomplextothetipsomewhatdiffersfromearlier
reports[26]andfromoriC–tipdistance(1.4±0.4μm,seeabove).Thisispossiblyduetodiffer-
entsampleprocessinganddataanalysis(useofcellwallstaininginsteadoftransmittedlight
images(Figs1,2and3)presumablyaffectstheanalysisofthehyphaltipposition).Thevaria-
tionofthetip-proximalParBcomplexpositioningintheparAmutantstrainswasveryhigh
(shownwithanF-test,theratioofvariances3.0±0.8forΔparA,3.2±0.7forparAoverproduc-
tionand1.7±0.4forparA strain(p<0.001))(Fig4D).
mut
OverexpressionofparAresultedinthemostnotablechangeoftheParBcomplexposition.
ImmunostainingofParAinthewildtypeparB-egfpstrainrevealedthattheParBcomplexwas
positionedattheedgeoftheParAsignal(Fig4B).TheoverproductionofParAledtoahuge
accumulationofParAatthehyphaltipsandshiftedtheParBcomplexawayfromthetip.
WeshowedbeforethattiplocalizationofParAisdependentonScyandParA protein
mut
ismislocalized[29].However,sincetheParA proteindoesnotinteractwithboth,ParB
mut
andScy,onthebasisofparA strainanalysis,wecannotconcludethattheScydependent
mut
localizationofParAdetermineslocalizationofParBcomplex.TheanalysisofParB-EGFP
fociintheΔscy,parB-egfpstrain(BD05)showedthatintheabsenceofScy,thedistance
betweenthetipandtheapicalParB-EGFPfocusinthenewlyformedbrancheswasmuch
morevariedthaninthe“wildtype”strainJ3310(S7Fig).Thisreinforcedthenotionthat
Scy-ParAinteractionisrequiredtoestablishanchoragefororiC/ParBcomplexsoonafter
branchemergence.
Finally,weexaminedwhetherParAinfluencestheoverallorganizationofthetipchromo-
somethroughtheParBcomplexandoriCanchorage.Toaddressthisquestion,weanalyzed
thelocalizationofParBcomplexesinconjunctionwithDNAstaining(Fig4C).Inthewild
typeparB-egfpstrain,theParBcomplexwasfoundatthetip-proximaledgeofthestained
nucleoid,whereasintheΔparAstrain,theParBcomplexwaslocatedfurtherawayfromthetip
thantheedgeofthenucleoid.MeasurementandplottingoftheParBfluorescenceandDNA
stainingintensitiesconfirmedthatintheabsenceofParA,thetip-proximalorientationofthe
oriC/ParBcomplexattheedgeofthenucleoidwaslost(Fig4E).
Torecapitulate,analysesoftheparAmutantstrainsrevealedthatthedistancebetweenthe
firstParBcomplexandthetipisdependentonParA.ParAinteractionswithScyandParB
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 8/25
ChromosomeSegregationduringHyphalGrowth
Fig4.LocalizationofParBcomplexisdependentonParA.(A)ImagesofParB-EGFP(green)complexesinthehyphaeof”wildtype”
parB-egfp(J3310)ΔparAparB-egfp(J3318),parA parB-egfp(DJ532),parA parB-egfp(DJ598)strains,mergedwithcellwall
overexp mut
staining(gray).(B)Co-localizationofParB-EGFP(green)withimmunostainedParA(blue)in“wildtype”parB-egfp(J3310)andparA
overexp
parB-egfp(DJ532).ToppanelshowstheParAimmunofluorescence(blue)mergedwithParB-EGFPfluorescence(green).Thebottom
panelshowsParB-EGFPfluorescence(green)mergedcellwallstaining(grey).(C)LocalizationofParB-EGFP(green)withinthenucleoid
(DNAstaining—red)in”wildtype”parB-egfp(J3310)andinΔparAparB-egfp(J3318).InpanelsA,BandCasterisksindicatethetipof
hyphaeandscalebars—1μm.(D)Thedistribution(shownasprobabilitydensityfunction)ofthedistancesbetweenthehyphaltipandthe
tip-proximalParB-EGFPcomplexin„wildtype”parB-egfp(J3310),ΔparAparB-egfp(J3318),parA parB-egfp(DJ532),parA parB-
overexp mut
egfp(DJ598)(analyzedfor170–300hyphae).(E)FluorescenceintensityofParB-EGFPandDNAstainmeasuredfromthehyphaltipin“wild
type”parB-egfp(J3310)andΔparAparB-egfp(J3318)(15and21hyphaeanalyzed).Foreachhypha,thefluorescencesignalwas
normalizedsothatthemaximumsignalwas100%.LinesaremodelsfittedusingaLoessalgorithmimplementedintheRprogram,greyarea
indicates95%confidenceinterval.DashedlineshowsmaximumParBfluorescenceintensityascalculatedbythemodel.
doi:10.1371/journal.pgen.1006488.g004
arepresumablyrequiredtoestablishthechromosomaltipanchorage.Furthermore,theParB
complexispositionedattheedgeofthetipParAassembly,andtheinteractionbetweenthe
ParBcomplexandParAorientatesthenucleoidwiththeoriCregiontowardthetipof
hyphae.
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 9/25
ChromosomeSegregationduringHyphalGrowth
AfterreplicationParAanchorsatthetiponeofthenewly–replicatedoriC
WehavedemonstratedthatParA,presumablythroughinteractionwithScy,anchorstheoriC/
ParBcomplexatthetip,assuringthatitfollowstheextendinghyphaltip.Thisraisestheques-
tionhowthisanchorageisestablishedafteroriCduplication.Toanswerthisquestion,weused
astrainwiththeEGFPtaggedreplisomes(DnaN-EGFP,J3337,[28])astheparentstrainfor
theintroductionoftheFROSsystem,tomonitorduplicationofthetipproximaloriCbytime-
lapseanalysis.
WesetouttoexaminehowdaughteroriCsbecomeanchoredafterreplication.Possiblesce-
nariosincludedalossofconnectionwiththetipduringoriCreplication;acloseassociationof
bothnewlyreplicatedoriCswiththetip,followedbyalossofanchorageofoneofthedupli-
catedoriCs;oranchorageofonlyoneofthenewlyreplicatedoriCstothetip.Byanalyzingthe
time-lapseimagesoftheFROSstrainexpressingdnaN-egfp(strainAK122),weobservedthe
appearanceofaDnaNfocusclosetoatip-proximalFROSfocus(Fig5Atoppanel,B,S8Fig,
toppanel,S2Movie).10minafterthereplisomeappearance,wedetected,bothnewlyrepli-
catedoriCsseparatedbyadistanceofaround0.8μminalmost15%ofthehyphae.Twenty
minuteslater,duplicatedoriCsseparatedbyadistanceofroughly1.3μmwerevisiblein
approximately70%ofhyphae(Fig5B,5Dand5E).However,thedistancebetweenthetipand
tip-proximaloriCwasthesame20–10minutesbeforeand10–20minutesafteroriCduplica-
tion(Fig5C).Thus,afterduplication,thetip-proximaldaughteroriCdidnotmovetowardthe
tip,butmaintainedaconstantdistancetothetip(Fig5Band5C).Interestingly,thesecondof
thenewlyreplicatedoriCsremainedco-localizedwiththereplisomeinthesubsequenttime-
lapseimages(Fig5Atoppanel,Fig5B,S8Fig,toppanel).Thedistancebetweenthetipand
tip-distaldaughteroriCsincreasedashyphaeextended(Fig5Band5C).Theseresultsindicate
thatwhiletiptheproximaloriCfollowsthetip,thesecondoriCstaysbehindtheextendingtip.
IntheΔparAFROSdnaN-egfpstrain(AK123),bothdaughteroriCsremainedcloselyassoci-
atedafterduplication(Fig5Abottompanel,S8Fig,bottompanel,S3Movie).IntheΔparA
strain(AK123),wedetecteddoubleFROSfocilaterafterreplisomeappearancethanobserved
inthe“wildtype”AK122.DaughteroriCsseparatedbyadistanceofapproximately0.9μm
werevisible20minutesafterreplisomeappearanceinonly12%ofthehyphae(Fig5Abottom
panel,Fig5Brightpanel,D,E).Approximately30minutesafterreplisomedetection,theaver-
agedistancebetweentheduplicatedoriCsreached1μminabout35%ofhyphae,butitdidnot
exceedthisvalue(forcomparison,inthewildtypehyphaeatthistimepointthedistancewas
1.3μmin70%ofhyphae).Interestingly,intheΔparAstrain,bothoriCsandreplisomesstillfol-
lowedtheextendinghyphaltip,butthedistancebetweenthefirstoriCandthehyphaltip
remainedgreaterthanthatfoundforthe“wildtype”AK122strain(Fig5C).Thisobservation
indicatesthat,althoughbothnewlyduplicatedoriCsstillmovebehindtheextendingtipinde-
pendentlyofParA(inΔparAstrain),theirseparationislessefficientandthefirstoriCisnot
attachedtothehyphaltip.
SinceParBcomplexesformateachoriCinthemultigenomichyphalcompartmentwe
expectedthatshortlyafterreplication,bothdaughteroriCsshouldbeboundbyParB.Totest
thisweusedthestrainexpressingparB-egfpanddnaN-mcherry(AK101).Time-lapseanalysis
showedthatParBcomplexduplication,asoriCduplication,wasdetected30minafterrepli-
someappearanceatthetipinalmost70%ofhyphae(S9Fig).
InordertoconfirmthatsegregationofduplicatedoriCinhyphaeisefficientinthepresence
ofParAwealsocheckedhowthenewlyreplicatedtip-distaloriCsareseparatedinthehyphal
stem.TheanalysisofthedistancesbetweenFROSfociaftertheirduplicationshowedthatthe
separationoftip-distalfociismuchlessefficientthanseparationoftip-proximalfociwhich
occursinpresenceofParA(S10Fig).
PLOSGenetics|DOI:10.1371/journal.pgen.1006488 December15,2016 10/25