Driving apart and segregating genomes in Archaea

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by  Barilla, Daniela

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TIMI 1355 No. of Pages 11
Review
Driving Apart and Segregating
Genomes in Archaea
Daniela Barillà1,*
Genomesegregationisafundamentalbiologicalprocessinorganismsfromall
Trends
domains of life. How this stage of the cell cycle unfolds in Eukarya has
Considerable diversity is observed in
been clearly defined and considerable progress has been made to unravel
thefeaturesofthecellcycleofdifferent
chromosome partition in Bacteria. The picture is still elusive in Archaea. The membersoftheArchaeadomain.
lineagesofthisdomainexhibitdifferentcell-cyclelifestylesandwide-ranging
Forthespeciescharacterizedtodate,a
chromosome copy numbers, fluctuating from 1 up to 55. This plurality of
markeddividehasemergedbetween
patternssuggeststhatavarietyofmechanismsmightunderpindisentangling monoploidCrenarchaeaandpolyploid
Euryarchaea with regard to chromo-
and delivery of DNA molecules to daughter cells. Here I describe recent
somecopynumber.
developments in archaeal genome maintenance, including investigations of
novel genome segregation machines that point to unforeseen bacterial and SMCproteinsappeartoplayanimpor-
tantroleinchromosomesegregation,
eukaryotic connections.
althoughfurtherstudiesareneededto
substantiatetheiractionandimpacton
theprocess.
Archaea: The Third Domain of Life
Archaeaarethethirdbranchofthetreeoflife[1].Sincetheirdiscovery40yearsago,membersof OrthologsofbacterialParADNAparti-
this [3_TD$DIFF]domain have been isolated from a vast array of diverse ecological niches, including soil, tion proteins are widespread across
Archaeaandhavebeenshowntobe
ocean plankton, freshwater lakes, acidic hot springs, volcanic mud, deep-sea hydrothermal
involvedinchromosomesegregationin
vents,andsaltylakes.Thefirstarchaeatobeanalyzedwerefromextremeecosystems,butthey synergywitharchaea-specificfactors.
arenowknowntobeubiquitousonourplanet.Forexample,ithasbeenestimatedthattheworld
ocean contains approximately 1.3 x 1028[2_TD$DIFF] archaeal cells [2]. Their ubiquity and abundance Recentinvestigationshavediscloseda
three-componentgenomesegregation
suggest that archaea are key players in regulating global biogeochemical cycles on Earth.
machinery borrowing building blocks
Archaea[1_TD$DIFF]havegeneratedalsoconsiderableinterestbecauseoftheirabilitytoadapttolifeunder fromBacteriaandEukarya.
extremeconditions,includinghighandlowtemperatures,veryacidicandalkalinepH,andhigh
salinity.
Sequencesavailableforanincreasingnumberofgenomes(521todate)togetherwithgenetic
andbiochemicalstudieshaveshownthatArchaeaexhibitamosaicoffeaturesfromtheother
twodomainsoflife,Bacteria(e.g.,energygeneration,metabolism,transport,nitrogenfixation
andCRISPR-cassystems)andEukarya(e.g.,DNAreplication,transcription,translation,and
proteinfolding).However,archaeaarealsocharacterizedbyuniquemolecularfeaturessuch
as methanogenesis and ether-linked isoprenoid lipid chains in their cell membranes [3].
Archaeaarealsointerestingforstudiesontheoriginoflife:thesemicrobescanbeconsidered
a‘timecapsule’thatprovidesaglimpseofwhatlifemayhavebeenlikeonEarthwhenthiswas
aplanetburstingwithgeologicalactivitybillionsofyearsago.MembersoftheArchaeadomain
that have been studied to date fall into three main phyla: Crenarchaea, Euryarchaea and
Thaumarchaea.
Despitetheprogressmadeindecodingmolecularmechanismsintheseorganisms,verylittleis 1DepartmentofBiology,Universityof
known about how the process of DNA segregation is organized in archaea and the subject York,YorkYO105DD,UK
remainsablackboxinthisdomainoflife.Thisreviewfocusesonrecentdevelopmentsinthearea
of archaeal genome segregation, discussing molecular machineries that have been recently
*Correspondence:
identifiedandemergingtrends. [email protected](D.Barillà).
TrendsinMicrobiology,MonthYear,Vol.xx,No.yy http://dx.doi.org/10.1016/j.tim.2016.07.001 1
CrownCopyright©2016PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
TIMI 1355 No. of Pages 11
Genome Organization: Chromosomes, Megaplasmids, and Smaller Replicons
Like bacteria, archaea are prokaryotic cells whose genetic material is not confined by a
membraneintoaseparatecompartment.Archaealgenomesconsistofacircularchromosome
andoftenalsolargeorsmallextrachromosomalelements.VirtuallyallthehalophilicEuryarchaea
sequencedtodateharboura2.0–3.9Mbpchromosomeandmultiplelargeplasmids[4].For
example,Haloarculamarismortuicontainsa3.13Mbpchromosometogetherwitheightaddi-
tionalrepliconsofwhichthelargest,pNG700,is410kbp.Manyspeciesarecharacterizedbya
dynamic flux between chromosome and plasmids which is facilitated by the presence of
numerous insertionsequences thatleadtoepisodesofintegrationofplasmidsintothechro-
mosome [5]. Accessory replicons also are found in Crenarchaea in the form of cryptic and
conjugativeplasmids,suchaspNOB8ofSulfolobusNOB8H2.However,crenarchaealplasmids
tend to be relatively small, with sizes below 50 kbp [6]. Unlike Bacteria that contain chromo-
somes with a single replication origin, and instead similarly to Eukarya, Archaea harbour a
chromosome containing oneor morereplicationorigins [5,7–9].
Cell Cycle in Archaea: Different Strokes for Different Folks
Everycellgoesthroughdefinedfunctionalstagesinthecourseofitslifespan,duringwhichvital
processes,suchasgrowth ofcellular structures,chromosomereplicationfollowedby segre-
gationanddivision,takeplaceinanorderedtimeline.Thestartandendpointsmarkthebirthof
newdaughtercells,andthelengthofinterveningtimedefinesthegenerationordoublingtime.
Thecellcycleinbacteriacomprisesthreestages:thegrowthorBphaseduringwhichthecell
activelysynthesisesproteins,lipids,andotherbuildingblocksinpreparationforDNAduplication;
thechromosomereplicationorCphase;andthepostreplicationorDperiodthatterminateswith
celldivision.Adifferentterminologyisadoptedfortheeukaryoticcellcyclewhichconsistsofthe
G1(gap1),S(DNAsynthesis),G2(gap2)andM(mitosis)stages.Thislatternomenclaturehas
been most commonly used to describe the cell cycle of members of archaeal phyla. The
knowledge built up so far indicates that archaea belonging to different lineages exhibit great
variability intheir cellcycle.
Crenarchaea
Pioneering work by the Bernander group in the 1990s initiated a survey of the cell cycle in
Crenarchaea.TheseinvestigationsrevealedthatmembersofthethermophilicgenusSulfolobus,
such as Sulfolobus solfataricus (doubling time (cid:1)425minutes) and Sulfolobus acidocaldarius
(doubling time (cid:1)213minutes), are characterized by a brief G1 prereplication period that
accounts for no more than 5% of the entire cell cycle [10]. The G1 ends with the inception
ofchromosomereplication,Sstage,whichproceedsfor30–35%ofthecellcycleandisfollowed
byaveryprotractedG2phase.Thispostreplicativeintervaloccupiesmorethan50%ofthecycle
andisadefininghallmarkofthecrenarchaealspeciesinvestigatedsofar[10,11].Membersofthe
genusSulfolobusaremonoploid:thecellsharbouronesinglechromosomeinG1stageand,
uponreplication,twocopiesarepresent.AstheG2stageisveryprolonged,Sulfolobusspecies
containtwochromosomecopiesformostofthecellcycle.DuringtheG2phasechromosomes
becomeorganizedforsegregation.Interestingly,bothcytologicalandbiochemicalstudieshave
indicatedthatthetwochromosomesremainpairedandconnectedforaprolongedtimeduring
G2phaseandappearasasinglenucleoidinmostcells[12,13].Afterwards,duringtheMphase,
chromosomesegregationtakesplaceveryswiftly,followedinrapidsuccessionbycelldivision.
Chromosomesegregationandcytokinesisoccurinatimespanequivalentto(cid:1)10%ofthecell
cycle andappearclosely interlinked.
Analogous cell-cycle patterns and timing have been observed for all the Crenarchaea spp.
studied to date which include Sulfolobus tokodaii, Acidianus hospitalis, Aeropyrum pernix,
Pyrobaculumaerophilum,andPyrobaculumcalidifontis[11].Althoughthenumberofcrenarch-
aeal species characterized so far is limited, interestingly all are monoploid and harbour two
2 TrendsinMicrobiology,MonthYear,Vol.xx,No.yy
TIMI 1355 No. of Pages 11
chromosomecopiesonlyoncompletionofDNAreplication(Figure1).Thisobservationsuggests
thatanaccurateandrigorousgenomesegregationmechanismmustoperateinthesespeciesto
ensure the faithful distribution of chromosomes to daughter cells. Whereas chromosome
segregation in bacteria occurs concomitantly with replication, the picture in Crenarchaea is
verydifferent:thetwoprocessesaretemporallyseparatedandgenomesegregationtakesplace
onlyattheendoftheprotractedG2stage[12,13].ThetwoPyrobaculumspp.thathavebeen
examinedrepresentaslightdeviationfromthecanonicalparadigmaschromosomesegregation
appears tobelargelysynchronized with replication [11].
Euryarchaea
TheEuryarchaeaphylumincludesawiderangeoffamiliesthatpopulatethemostdiverseniches
andexhibitdisparatelifestyles.Thesulphate-reducingArcheoglobusfulgidusdisplayscell-cycle
features resemblingthose observedforcrenarchaeal Sulfolobusspp.[14].
Incontrast,themethanogenMethanocaldococcusjannaschiiispolyploidandcharacterizedby
a very relaxed cell cycle [15]. Cells in exponential phase accommodate between 3 and 15
chromosomecopiesthatarereducedtoanumberbetween1and5instationaryphase.Cell
division occurs asymmetrically, resulting in an uneven distribution of chromosome copies to
daughtercells[15].Theapparentrandomnessandlackoforderdominatingtheseprocesses
beg the question of whether a genome segregation system operates in M. jannaschii and
whether the presence of multiple chromosome copies makes a DNA-partitioning apparatus
dispensable.Anothermethanogenbelongingtoadifferentorder,Methanothermobacterther-
mautotrophicus,growsaschainsofrod-shapedcellseachofwhichcontainstwochromosomes
Polyploid Diploid Monoploid
Euryarchaea
Halobacterium salinarum
Haloferax volcanii
Archeoglobus fulgidus
Thermococcus kodakarensis
Pyrococcus furiosus
Methanocaldococcus
jannaschii
Methanococcus
maripaludis
Methanothermobacter
thermautotrophicus
Crenarchaea
Acidianus hospitalis
Sulfolobus solfataricus
Sulfolobus acidocaldarius
Sulfolobus tokodaii
Pyrobaculum aerophilum
Pyrobaculum calidifon(cid:2)s
Figure 1. Ploidy in a Set of Characterized Members of the Euryarchaea and Crenarchaea Phyla.Allthe
euryarchaealspecies(greenbox)containmultiplechromosomecopies,whereasthecrenarchaeal species(bluebox)
harbourasinglechromosome.
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TIMI 1355 No. of Pages 11
intheG1phasewhichsegregatesoonafterreplication[16].InthiscasetheG2stageisverybrief
or completely absent. Methanococcus maripaludis is an extreme example of polyploid eur-
yarchaeon with asmanyas55 chromosomecopies [17].
Halophilic archaea are characterized by the presence of multiple copies of the chromosome
(Figure 1). To some extent this feature has hindered a detailed dissection of their cell cycle.
Halobacterium salinarum contains approximately 30 chromosome copies during exponential
phasewhicharereducedtoaround10instationaryphase[18].Interestingly,thisarchaeondoes
nothaveatemporallydemarcatedSstage,asDNAreplicationoccursthroughoutthecellcycle
[19].However,lackofatightreplicationcontroldoesnotresultinaderegulatedcellcycle,as
shownbythecompleteblockofcelldivisionuponinhibitionofDNApolymerase[20].Further-
more,genomesegregationmechanismsthatdeliverequalnumberofchromosomestothetwo
daughtercellsappeartobeinplaceinH.salinarum[18].Adifferenthalophile,Haloferaxvolcanii,
is also highly polyploid, with cells harbouring approximately 20 chromosome copies in expo-
nential phase and approximately 12 during stationary phase [18]. The chromosome copy
numberoftheeuryarchaeonThermococcuskodakarensishasalsobeenrecentlyinvestigated.
Analogously to the situation in other members of this phylum, T. kodakarensis cells show
polyploidywithachromosomecopynumberfluctuatingbetween19and7fromexponentialto
stationary phase[21].
Assummarizedabove,allEuryarchaeainvestigatedtodatearepolyploid,withtheexceptionof
M. thermautotrophicus that is diploid. This is in sharp contrast with observations related to
characterized Crenarchaea which are all monoploid (Figure 1) [17]. Accommodating and
managing multiple chromosome copies raises a number of interesting biological questions,
including the mechanism of DNA packaging within relatively small cells. A recently proposed
hypothesissuggestsadirectcorrelationbetweenthepresenceofhistonesandpolyploidy[21].
In fact,histones arecommonly foundin Euryarchaea,but notin Crenarchaea [22,23].
Thaumarchaea
TheThaumarchaealineagewasrecognizedasanindependentphylumin2008anditsmembers
are widespread in terrestrial and ocean niches [24]. The cell cycle of the ammonia-oxidizer
NitrosopumilusmaritimusshowsaprereplicationG1phasethatislongerthanthatobservedfor
Crenarchaeaandisequivalentto19–29%ofthefullcellcycle.AveryprotractedSstagefollows,
whichcorrespondsto45–53%ofthecycle:remarkably,15–18hoursarenecessarytoreplicate
thechromosome.Genomesegregationoccursquicklyafterreplicationtermination,withtheG2
phase beingvery shortor absent[25].
Altogether,thestudiesonthearchaealcellcycleconductedsofarhavehighlighteddisparities
andanalogiesamongphylaand,interestingly,adichotomybetweenmonoploidandpolyploid
archaeahasemerged(Figure1).Cellsharbouringasinglechromosomecopyneedtoemploya
segregationmechanismtoensureanaccuratedistributionofthegeneticmaterialinheritedby
theprogeny.Thepresenceofmultiplecopiesofthechromosome,sometimesasmanyas55as
inMethanococcusmaripaludis[21],raisesthequestionastowhetherpolyploidarchaearequire
a chromosome-sorting partition machine.
Delivering Genomes to Daughter Cells: Snapshots from Bacteria
The moleculareventsandfactorsunderpinningchromosomesegregationin eukaryoteshave
been extensively investigated. During mitosis, the microtubules of the mitotic spindle capture
sisterchromatidsandpullthemtooppositespindlepoles[26].Themechanismsandproteins
that drive chromosome segregation in bacteria are not fully elucidated; however, significant
progresshasbeenmadeinthepasttwodecades.Interestingly,onlyafewcomplexeshavebeen
identifiedaskeyplayersin the process ofbacterial DNAsegregation.
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TIMI 1355 No. of Pages 11
Moving DNAMolecules Apart: The [4_TD$DIFF]ParABS System
PioneeringworkbytheAustinandHiragagroupsidentifiedtheParABmoduleasresponsiblefor
theactivepartitionoflow-copy-numberplasmidsinEscherichiacoli[27,28].Thissystemwas
laterfoundtobeencodedbymostbacterialchromosomes[29]andconsistsofthreecompo-
nents:twoproteins,ParAandParB,inadditiontoacis-actingcentromere-likeparSsite[30].
ParAisaWalker-typeATPasethatinteractswithParBandnonspecificDNA,whereasParBisa
site-specific DNA-binding protein, which recognizes and associates with the parS site. Once
boundbyParB,low-copy-numberplasmidsarecapturedbyParAthat,throughcyclesofATP
binding and hydrolysis, forms dynamic patterns on the nucleoid, in this way moving and
eventually positioning sister plasmids in diametrical opposite locations of the dividing cell
[31]. ParAB systems encoded by chromosomes are involved in the segregation of newly
duplicated origins of replication (oriC), adopting dynamics analogous to those described for
plasmidsandtranslocatingtheoriginstowardsoppositecellpoles[32].Recentinsightsintothe
roleofParABinchromosomesegregationhavebeenprovidedparticularlybystudiesonVibrio
cholerae[33],Caulobactercrescentus[34–38],Bacillussubtilis[39,40],Pseudomonasaerugi-
nosa [41,42],Streptomyces coelicolor[43], andMyxococcusxanthus [44].
Organizing the Chromosome forSegregation: SMCCondensin
Structural maintenance of chromosomes (SMC) proteins are conserved across the three
domains of life and mediate crucial chromosome biology processes such as condensation,
sisterchromatidcohesion,segregation,andDNArepair[45].Thegenomesofmostbacteriaand
archaeaharbourasinglesmcgene[46].MostbacterialSMCproteinsformacomplexwithtwo
otherfactors,ScpAandScpB.Thisassemblyisalsoreferredtoascondensinandplaysakey
role in compacting the chromosome by bridging and interconnecting DNA loops [32]. SMC
condensins are recruited to the bacterial chromosome oriC region via interaction with ParB
boundtotheparSsitesthatareclusteredaroundtheoriginofreplication[39,40].Thisorganized
gathering of condensins at the origin imparts a particular structure to the chromosome and
mediates its segregation. Very recent studies have shown that condensins act as molecular
‘staples’that align chromosomearms in closeproximity[47,48].
Genome Segregation in Archaea: Potential Suspects and Identified Players
Instarkcontrastwitheukaryotesandbacteria,ourknowledgeonchromosomesegregationin
archaea is very rudimentary, partly due to the fact that most archaeal genomes have been
sequencedonlyinthelastdecade,butalsoattributabletothedevelopmentofgenetictoolsto
manipulate some archaea only in recent years. [5_TD$DIFF]I report the findings of the few investigations
conducted so far,discussingimplications andquestions that still needto beaddressed.
The Roleof SMCCondensins
SMCcondensinsarewidespreadacrossarchaealphyla.Earlystudiesanalysedthepossible
involvement of the SMC protein of Methanococcus voltae in chromosome segregation.
Inactivation of the smc gene in this euryarchaeon resulted in aberrant genome partition
and cell morphology [49]. Approximately 20% of the cells harboured no chromosome, and
around 2% displayed a size that was three to four times larger than that of wild-type cells.
Quantitation of the DNA of these so-called titan cells showed a content 10–20-fold higher
thanthatpresentinnormalcells.ThisphenotypeindicatesthattheSMCproteinpresidesover
an important cell-cycle checkpoint in M. voltae and plays a crucial role in chromosome
segregation [49].
Soppaandcolleaguescharacterizedthecell-cycleprofileofanSMC-likeprotein,namedSph1,
inH.salinarum[19].Usingsynchronizedcultures,sph1geneexpressionwasshowntobecell-
cycle-regulatedwithapeakatthestageofcelldivisionseptumformation.Giventhatmaximal
expressionisreachedatalatestageofthecellcyclewhenchromosomesegregationisnearly
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TIMI 1355 No. of Pages 11
completed,Sph1mightbeinvolvedinDNArepairinafinalstepofchromosomereplication[19].
Whether this SMCfactorhas arolein genome segregation remainstobeelucidated.
Despitethepaucityofinformationthusfar,SMCproteinsareanticipatedtoplayasignificantrole
inchromosomesegregation,basedonthehighlevelofconservationandbyanalogywiththe
mechanisms uncovered inbacteria.
The SegAB System:A Hybrid DNA-Partition Machine
Arecentstudyhasreportedtheidentificationandinitialcharacterizationofadedicated chro-
mosome-segregation system in the thermophilic crenarchaeon S. solfataricus [50]. This
genome-partitioning apparatus consists of two proteins, SegA and SegB, and a cis-acting
centromere-likeregion(Figure2A).Intriguingly,thecomplexisahybridpartitionmachine:SegA
isanorthologofbacterial,Walker-typechromosome-encodedParAproteins,whereasSegBis
an archaea-specific factor lacking any sequence identity to either eukaryotic or bacterial
proteins.However,SegBdisplayssequenceidentitytoagroupofconserved,uncharacterized
proteinspresentinbothCrenarchaea((cid:1)80%identity)andsomeEuryarchaea(30–46%identity)
(Figure2B).Interestingly,thegenesencodingSegBproteinsarelocatedinvariablydownstream
ofsegAorthologs.BLASTsearchesagainstarchaealgenomesavailablesofarindicatedthatthe
segABcassetteispresentinanarrayofarchaealgenerabelongingtobothCrenarchaeaand
Euryarchaea phyla (Figure 2B). Although the ploidy and genome content has not been
(A)
sso0033 segA segB
(B) Thermococcus litoralis Euryarchaea
Thermococcus sibiricus
Methanosphaera stadtmanae
Methanothermus fervidus
Methanobrevibacter ruminan(cid:2)um
Methanobrevibacter smithii
Methanothermobacter marburgensis
Methanothermobacter thermautotrophicus
Methanobrevibacter sp. AbM4
Methanobacterium sp. AL–21
Methanobacterium sp. SWAN–1
Methanobacterium formicicum
Sulfolobus islandicus HVE10/4 Crenarchaea
Sulfolobus islandicus Y.G.57.14
Sulfolobus islandicus M.14.25
Sulfolobus islandicus M.16.4
Sulfolobus solfataricus P2
Sulfolobus tokodaii
Sulfolobus acidocaldarius
Acidianus hospitalis
Sulfolobales archaeon AZ1
Sulfolobales archaeon Acd1
Metallosphaera sedula
Figure2.TheSegABSystemIsWidespreadacrossArchaea.(A)OrganizationofthesegABcassette,includingthe
upstreamsso0033geneandthetwoDNAsites(inlilac)towhichSegBbinds.(B)Phylogenetictreeofanonexhaustivesetof
SegB orthologs. Genomic context studies show that each segB gene is accompanied by a segA gene. Blue box,
crenarchaeal SegB cluster; green box, euryarchaeal SegB orthologs. Within the crenarchaeal cluster the Sulfolobus
solfataricusP2strain,whoseSegABhavebeencharacterized,isshowninred.
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TIMI 1355 No. of Pages 11
determined for all these genera, what is tantalizing is that the majority of these Archaea are
monoploid,oratmostdiploidsuchasM.thermautotrophicus.Ifacellharboursonlyoneortwo
copiesofthechromosome,thenarigoroustoolkittosegregateDNAatcelldivisionisastringent
sinequanon.The30endofsegAoverlapswiththe50endofsegB:thisarrangementsuggests
thatthegenesmaybepartofasingletranscriptionalunitimplyingthatSegAandSegBwork
togethertoeffectthesamebiologicalprocess.Supportingevidencederivesfromatranscription
profilingstudyshowingthattheSulfolobusacidocaldariushomologuesofsegAandsegBare
coexpressed in acell-cycle-regulated fashion[51].
SegA is an ATPase assembling into higher-order structures in vitro upon ATP binding, while
SegB is a site-specific DNA-binding protein contacting palindromic sequences located
upstream of the segAB cassette [50]. The two proteins interact with one another, and SegB
synergistically affects SegA self-assembly dynamics, perhaps acting as a nucleator protein.
SegB is a dimeric protein that binds specifically to an imperfect palindromic motif located
upstreamofthesegAstartcodon(site1)andthenatposition–59withrespecttothesamestart
codon(site2)(Figure2A)[50].Thesesitesmightbearchaealcentromereanalogs.However,at
thisstageitcannotberuledoutthatthesitesmightalsoactasregulatoryregionsthatcontrolthe
expressionofthesegABcassette.Whetheradditionalsitesarescatteredacrossthechromo-
some iscurrently unknown.
MicroscopyinvestigationshaverevealedthatincreasedexpressionofsegABinS.solfataricus
cellsdisruptschromosomesegregation,asevidencedbythepresenceofanucleatecells,highly
condensednucleoidssqueezedintoone-halfofthecellvolumeandsplit,guillotinedchromo-
somes [50]. These findings indicate that SegA and SegB play a key role in chromosome
segregation. Further support comes from the observations that segAB are highly repressed
uponUVirradiation[52]andthattheirexpressionstartsconcurrentlywiththeinitiationofDNA
replication[51],bothofwhichunderscoreafunctioninchromosomesegregation.Themecha-
nism underpinning how the SegAB complex drives sister chromatids apart remains to be
elucidated.
The AspA–ParBA Machinery: Borrowing Building BlocksfromBacteriaandEukaryotes
Sulfolobus NOB8H2 is a strain isolated by the archaea pioneer WolframZillig and coworkers
fromacidichotspringsatNoboribetsuintheislandofHokkaido,Japan[53].Thisstrainharbours
a41kbpconjugativeplasmid,pNOB8,whosesequencehasbeendetermined[54].Theplasmid
contains (cid:1)50 ORFs including two tandem genes, orf45 and orf46, whose products show
homology, respectively, to ParB and ParA families of bacterial partition proteins. The 36 kDa
polypeptide encoded by orf46 is a 315-residue protein with similarity (33–37%) to bacterial
ParAs.orf45encodesa470-aminoacidprotein(55kDa),whosehomologytobacterialParBsis
confinedtotheN-terminaldomain(residues1–190)(42–58%similarity),whereastheC-terminus
shareshomologywitheukaryoticproteins,includingkinesin-likemotorproteins.Interestingly,a
closerinspectionoftheregionimmediatelyupstreamofparBrevealedasmallgene,orf44,which
encodesa93-aminoacidproteinof10.7kDawithnosequencehomologytoanycharacterized
segregationprotein[55].The30endofthisgeneoverlapswiththe50endofparB,andsimilarly,
the 30 end of parB overlaps with 50 end of parA (Figure 3A). This arrangement suggests that
orf44,parB,andparAmaybepartofasingletranscriptionalunit.Atricistronicpartitioncassette
isaninterestingfeaturethatisnotcommoninthebacterialdomain,whosetypicalsegregation
modulesarebicistronic[30,31].Furthermore,thereisevidencesuggestingthattheorf44–parBA
cassetteofthisplasmidencodesabonafidepartitionsystem:whenpNOB8istransferredby
conjugationintoadifferentSulfolobusstrain,theplasmidundergoesageneticrearrangement
duetoasinglerecombinationevent,whichproducesthedeletionvariantpNOB8-33[53,54].
This plasmid presents a deletion of a (cid:1)8 kbp region resulting in the loss of the orf44–parBA
cassette andisnotstably maintained[[6_TD$DIFF]53].
TrendsinMicrobiology,MonthYear,Vol.xx,No.yy 7
TIMI 1355 No. of Pages 11
(A) aspA parB parA
(C)
(B)
(D)
Figure3.OrganizationandStructuresofthepNOB8AspA–ParBASystem.(A)Schematicdiagramofthegene
cluster.The30endofaspAoverlapswiththe50endofparB,andthe30endofparBoverlapswiththe50endofparA.(B)
AspA–DNAstructure(PDB5FC0)showingthreeinteractingAspAdimers(inorange,green,andblue)associatedwiththe
DNAfragmentcontainingthe23bpputativecentromericsite.(C)Adenylyl-imidodiphosphate(AMP-PNP)-boundParA
dimerstructure(PDB4RU8).TheATPanalogAMP-PNPisshowninred.(D)(Left)ParB-Nstructure(PDB4RSF);(right)
ParB-Cdimerstructurewithonemonomershowningreenandtheotherinmagenta(PDB4RS7).Thestructuralimages
weregeneratedbyusingPyMOLversion1.8.0.7(Schrodinger)usingtheindicatedPDBcoordinates.
A very recent study has provided structural and mechanistic insights into this novel DNA
segregation machinery [55]. Orf44, renamed AspA (for archaeal segregation protein A), is a
dimeric, site-specific DNA-binding protein that recognizes a 23bp palindromic motif located
upstream of its gene. DNase I footprints have shown that AspA binds to the 23bp putative
centromereand,athigherconcentrations,spreadsontheDNAinthe50 direction,protecting
over200bpfromtheinitialnucleationsite[55].Incontrast,ParBbindsDNAnonspecificallyonly
athighconcentrations,whichrepresentsadeparturefromthebacterialparadigm.Thestructure
of AspA discloses an elongated dimer containing a winged helix-turn-helix DNA binding fold.
Remarkably, the AspA–DNA structures exhibit multiple AspA dimers (Figure 3B) that, when
extended by packing, lead totheassembly of acontinuous left-handed helix.
Interaction investigations established that pNOB8 ParB binds to AspA and ParA. However,
AspAdoesnotassociatewithParA,suggestingthatParBmightactasanadaptorproteinwithin
thecomplex.With470residues,pNOB8ParBislargerthancanonicalParBproteinsfoundin
bacteria,andconsistsoftwodistinctdomains,ParB-N(residues1–320)andParB-C(residues
370–470)connectedbyaflexiblelinker.AspAinteractswithParB-Nonly,whereasParAdoes
not bind to either the N- or C-terminus of ParB, suggesting that the extended linker region
contains the ParA contacting interface [55]. ParB-C was found to mediate nonspecific DNA
8 TrendsinMicrobiology,MonthYear,Vol.xx,No.yy
TIMI 1355 No. of Pages 11
binding.Thedeterminationofthethree-dimensionalstructurerevealedthatParB-Nsharesweak Outstanding Questions
similarity with the N-terminus of the chromosome segregation ParB, Spo0J, of Thermus Do polyploid archaea use an active
thermophilus (Figure3D, left) [55,56]. systemforchromosomesegregation?
If so, what is this system? If not, do
these microbes rely on a stochastic
Furthersmall-angleX-rayscattering(SAXS)studiesontheParB-N-AspAcomplexshowedthata diffusionprocess?
ParB-NdimerencasesthesidesandtopoftheAspAdimer.Interestingly,intheSAXSmodel,
ParB-NdimerscanbedockedontoeachAspAdimerintheAspA-DNAhelix,fittinginalock- Howdohalophilicarchaeahandlethe
and-keyfashionintothehelixgroovesandgeneratingamultiproteinsuperhelicalstructure[55]. simultaneous segregation of multiple
replicons that include chromosome
InthisassemblytheParB-CprotrudesintothesolventandisconnectedtotheParB-Ndomain
andmegaplasmids?
through the long flexible linker. As ParB-C binds nonspecific DNA, this domain is free to
associate with random DNA sequences on either the plasmid or chromosome, or both. Which and how many genome-parti-
Interestingly,thisactivityindicatesthatParBisnotsimplyanadaptor‘cushion’sittingbetween tionsystemsareencodedbydifferent
AspAandParA,butisinvolvedinadditionalaspectsofthesegregationprocess.Surprisingly,the archaea?
structureofParBC-terminusexhibitsafoldsimilartothatoftheCenpAhistonevariantwhich
How is genome segregation coordi-
replaceshistoneH3oncentromeresequencesandisinvolvedinassemblyofthekinetochore
nated with DNA replication and cell
segregation machinery in eukaryotic cells (Figure 3D, right) [57]. This unforeseen observation divisioninarchaea?
drawsaparallelbetweenDNAsegregationinarchaeaandeukaryotes.Afurthersignificanceof
thefindingliesinthat,todate,histonehomologshavebeenidentifiedinEuryarchaea;however, Doesthearchaealchromosomeadopt
aspecificorientationinrelationtoref-
theyare anexception in Crenarchaea.
erencepointswithinthecell?
ThestructureofpNOB8ParAshowsstrongresemblancetobacterialWalker-typesegregation
Whatarethecharacteristicsofcentro-
proteins, such as the chromosome ParA homolog, Soj, from Thermus thermophilus [58] and meres on archaeal chromosome and
multidrugresistantplasmidTP228ParAhomolog,ParF(Figure3C)[59].SimilartobacterialParA plasmids? Are the centromers clus-
proteins,pNOB8ParAshowsnonspecificDNA-bindingactivity[55].Thisfindingsuggeststhat teredinspecificpositionsordispersed
morerandomly?
ParA might bind the nucleoid in Sulfolobus NOB8H2 and thereby might allow anchoring and
transport of the plasmid with the aid of ParB. However, the mechanism underlying pNOB8
HowdoestheSegABcomplexmedi-
segregation remains tobeelucidated. ate chromosome segregation? How
conservedandwidespreadisthissys-
Altogether, the AspA–ParB–ParA complex is a novel three-component segregation machine, tem across Euryarchaea? Are there
variationsontheSegABthemeinCren-
encodedonbothcrenarchaealplasmidsandchromosomes,thatmergesbuildingblocksfrom archaeaandEuryarchaea?
bacteria and eukaryotes and opens exciting fresh perspectives on genome segregation in
archaea. Dosphericalarchaealcellshavefunc-
tional‘poles’?Ifyes,dothepolesplaya
roleinchromosomesegregation?
Concluding Remarks
Archaeaareubiquitousinhabitantsofourplanet.Theirabilitytothriveinnicheswherenoother
What is the mechanism underlying
organismcansurvivemakesthemremarkableobjectsofinvestigationforbasicstudiesonlife plasmid segregation mediated bythe
pushed to extremes, but also interesting microbes from which to harness molecules and AspA–ParBA assembly? Is pNOB8
resources for novel biotechnology applications. The recent identification of Lokiarchaea, a chimaeric ParB protein the prototype
of a novel family of crenarchaeal
newlyproposedphylumwithdistinctiveeukaryoticsignatures[60],hasrekindledthepassionate
histones?
debateontheoriginofEukarya.However,despite521sequencedgenomesandawealthof
molecularstudies,thefundamentalbiologicalprocessofgenomesegregationremainsaterra
still vastlyincognita.
Initialinvestigationsonthecellcycle,nucleoidmorphology,andchromosomecopynumberhave
laid the foundations for exploring genome partition and highlighted the spectrum of different
lifestylesadoptedbyarchaea,whenitcomestoarranging,condensing,anddispatchingtheir
chromosomes. The advent of next-generation sequencing and metagenomics as well as the
development of genetic tools to manipulate archaea have allowed the identification of genes
encodingcomponentsofpossibleDNA-segregationengines.Asobservedinotheraspectsof
archaealbiology,achimaericnatureseemstobeattheheartofrecentlycharacterizedgenome-
segregationmachineriesthatmergebacterialandeukaryoticelements.Howdothesearchaeal
complexespullDNAmoleculesapart?Andhowdotheseassembliescoordinatetheiractionin
TrendsinMicrobiology,MonthYear,Vol.xx,No.yy 9