from essential cell cycle control to ancillary functio

14 Pages·2015·1.54 MB·English

FEMSMicrobiologyReviews,39,2015,120–133
doi:10.1093/femsre/fuu002
ReviewArticle
REVIEW ARTICLE
Versatility of global transcriptional regulators in
alpha-Proteobacteria: from essential cell cycle control
to ancillary functions
Gae¨l Panis1, Sean R. Murray2 and Patrick H. Viollier1,∗
1DepartmentofMicrobiologyandMolecularMedicine,InstituteofGeneticsandGenomicsinGeneva(iGE3),
FacultyofMedicine/CMU,UniversityofGeneva,RueMichelServet1,1211Gene`ve4,Switzerlandand
2DepartmentofBiology,CenterforCancerandDevelopmentalBiology,InterdisciplinaryResearchInstitutefor
theSciences,CaliforniaStateUniversityNorthridge,18111NordhoffStreet,Northridge,CA91330-8303,USA
∗
Correspondingauthor:DepartmentofMicrobiologyandMolecularMedicine,InstituteofGeneticsandGenomicsinGeneva(iGE3),Facultyof
Medicine/CMU,UniversityofGeneva,RueMichelServet1,1211Gene`ve4,Switzerland;E-mail:[email protected];Tel.:[phone];
Fax:[phone]
Onesentencesummary:ThreetranscriptionalmodulescomposedofantagonisticregulatoryproteinsdirectcellcycletranscriptioninCaulobacter
crescentus,whilealsooftenperformingnon-essential,yetglobal,regulatoryfunctionsindiversealpha-proteobacteriallineages.
Editor:TamMignot
ABSTRACT
Recentdataindicatethatcellcycletranscriptioninmanyalpha-Proteobacteriaisexecutedbyatleastthreeconserved
functionalmodulesinwhichpairsofantagonisticregulatorsactjointly,ratherthaninisolation,tocontroltranscriptionin
S-,G2-orG1-phase.Inactivationofmodulecomponentsoftenresultsinpleiotropicdefects,rangingfromcelldeathand
impairedcelldivisiontofairlybenigndeficienciesinmotility.Expressionofmodulecomponentscanfollowsystemic(cell
cycle)orexternal(nutritional/celldensity)cuesandmaybeimplementedbyauto-regulation,ancillaryregulatorsorother
(unknown)mechanisms.Here,wehighlighttherecentprogressinunderstandingthemoleculareventsandthegenetic
relationshipsofthemodulecomponentsinenvironmental,pathogenicand/orsymbioticalpha-proteobacterialgenera.
Additionally,wetakeadvantageoftherecentgenome-widetranscriptionalanalysesperformedinthemodel
alpha-ProteobacteriumCaulobactercrescentustoillustratethecomplexityoftheinteractionsoftheglobalregulatorsat
selectedcellcycle-regulatedpromotersandwedetailtheconsequencesof(mis-)expressionwhentheregulatorsareabsent.
Thisreviewthusprovidesthefirstdetailedmechanisticframeworkforunderstandingorthologousoperationalprinciples
actingoncellcycle-regulatedpromotersinotheralpha-Proteobacteria.
Keywords:cellcycletranscription;alpha-Proteobacteria;Caulobactercrescentus;globalregulator;CtrA;GcrA;CcrM
methyltransferase;SciP;MucR;epigenetics
INTRODUCTION
centus (Laub et al., 2000). The long-awaited matching observa-
tions were recently made in the related alpha-proteobacterial
Fifteen years ago, the first evidence was provided that a vast
plantsymbiontSinorhizobiummelilotifromtheorderRhizobiales
numberoftranscriptsfluctuateasafunctionofcellcyclepro-
(DeNiscoetal.,2014).Togetherthesestudiesprovidestrongevi-
gressionintheaquaticalpha-ProteobacteriumCaulobactercres-
dencethattranscriptoscillationsmaybeageneralfeatureofthe
Received:7August2014;Accepted:13November2014
(cid:3)C FEMS2015.Allrightsreserved.Forpermissions,pleasee-mail:[email protected]
120
Panisetal. 121
Figure1.Cellcycleprogressionandconservationofglobaltranscriptionalregulatorscontrollingcellcycletranscriptioninalpha-Proteobacteria.A.Schematicofthe
C.crescentusandS.meliloticellcycles.CaulobactercrescentusandS.melilotidivideasymmetricallyattheendofeachcellcyclegivingrisetotwoprogenycellswith
distinctsizesandfates.ThesmallerG1-arresteddaughtercellresidesinaquiescentnon-replicative(G1-like)stateandmustdifferentiateintotheS-phasereplicative
formbeforeproceedingtodivision.WhilebothS.melilotidaughtercellshaveperitrichousflagellaandaremotile,onlyonedaughtercellisflagellatedandmotilein
C.crescentus.NotethatS.melilotihasthreereplicons(acircularchromosomeandtwolargeplasmids)butforsimplicityitsgenomeisrepresentedhereasonecircular
chromosomeasinDeNiscoetal.(2014).TheG1-arrestedCaulobacterdaughtercell,alsoknownas‘swarmer’cell,ismotileandchemotactic.Itpossessesseveral
adhesivepiliandasingleflagellumatonepole.Duringtheprocessofdifferentiationintothesessile‘stalked’cell,theflagellumandpiliarelostandreplacedbyatubular
stalkstructureharboringanadhesiveholdfastwhichiselaboratedfromthevacatedpole.B.Asdiscussedinthisreview,asetofglobaltranscriptionalregulatorspromote
theriseandfallofselectedtranscriptsinC.crescentusandthesecellcycleregulatorsareconservedamongthealpha-Proteobacteria.Thephylogenetictreeshowsthe
presence(greybox)orabsence(whitebox)ofgenespredictedtoencodeorthologsofDnaA,GcrA,CcrM,CtrA,SciPandMucR.ThisphylogenetictreeisadaptedfromFig.2
andtableS2ofBrillietal.(2010)forDnaA,GcrA,CcrMandCtrAorthologousgenedistributions.BLASTanalysistoolfromNCBI(http://blast.ncbi.nlm.nih.gov/Blast.cgi)
wasusedtodeterminethedistributionofSciPandMucRinthesameselectedalpha-proteobacterialdataset.
alpha-proteobacterial cell cycle and perhaps the cell cycles of thelargerdaughterresidesinS-phase,progressesintotheG2-
other bacterial phyla, irrespective of the ecological niche that phase transcriptional program of a pre-divisional cell. Upon
the bacteria occupy. As a plant symbiont, S. meliloti lives in compartmentalization of the pre-divisional cell into two un-
a different habitat (soil) than C. crescentus (fresh-water), but equallysizedchambers,theG1-phasetranscriptionalprogram
bothlineagesrelyonconservedsystemiccuesandeffectorpro- isre-instatedinthesmallerchamberandS-phasetranscription
teinstoorchestratetranscriptfluctuationswithcellcyclepro- resumesintheother(Fig.1A).
gression (Brilli et al., 2010; Ardissone and Viollier 2012). A cell In C. crescentus, the larger S-phase cell features a stalk at
cycle-regulated polysaccharide capsule permits synchroniza- theoldpoleanditmaturesintopre-divisional(G2-phase)cell
tionofC.crescentusculturesbysimpledensity-gradientcentrifu- thathasasingleflagellumandthepilussecretionmachineryat
gation(Ardissoneetal.,2014).Owingtothistechnicaladvantage thenewcellpole.AflagellatedandpiliatedG1-phase(swarmer)
alongwiththegenetictractabilityofC.crescentus,themolecu- cellandanon-motileS-phase(stalked)cellemergefromeach
larunderpinningsofcellcycletranscriptionwereprimarilydis- division (Skerker and Laub 2004). Forward genetics was used
sectedinthismodelsystem,butimportantrecentstudiesfrom to unearth several key factors regulating cell cycle transcrip-
alpha-proteobacterialsymbiontsandpathogenslendcredence tion (Fig. 2A). Bypass mutants (Murray et al., 2013; Fumeaux
totheideathatthefeaturesapplytomanyothersystems(Laub etal.,2014)revealedthattheidentifiedtranscriptionfactorsact
etal.,2000;DeNiscoetal.,2014;Degheltetal.,2014;Fumeaux inantagonisticpairs(definedhereastranscriptionalmodules)
etal.,2014). that direct cell cycle transcription sequentially to S-, G2- and
The idea that cell cycle-controlled transcription is likely G1-phase(Fig.2B).TheGcrAtranscriptionfactor(Holtzendorff
widespreadinalpha-Proteobacteriacouldbeinferredbytheob- etal.,2004)andtheCcrMadeninemethyltransferase(Zweiger,
servationthatcelldivisioninmanylineagesismorphologically MarczynskiandShapiro1994),whosemethylationattheN6po-
asymmetric(Hallezetal.,2004),yieldingdaughtercellsthatre- sitionofadenine(m6A)inthecontextof5(cid:4)-GANTC-3(cid:4) recogni-
side in different cell cycle stages in which distinct transcrip- tionsequenceindouble-strandedDNA(henceforthGANTC),are
tionalprogramsshouldbeactive(Laub,ShapiroandMcAdams requiredforefficienttranscriptionofmanyS-phasepromoters
2007; De Nisco et al., 2014; Deghelt et al., 2014). The smaller (Fioravantietal.,2013;Murrayetal.,2013;Gonzalezetal.,2014).
daughter cell is temporarily arrested in a quiescent G1-like Bycontrast,CtrA,aresponseregulatorthatalsonegativelyregu-
phase,unabletoreplicateitsgenomeortodivide.Bycontrast, latesreplicationinitiation(Quon,MarczynskiandShapiro1996)
122 FEMSMicrobiologyReviews,2015,Vol.39,No.1
Figure2.RefinementoftheC.crescentusregulatorynetworkorchestratedbythreetranscriptionalmodulesactingoncellcycle-regulatedtargetpromoters.A.Represen-
tationofthegeneticregulatorynetworkcontrollingcellcycletranscriptionasdetailedpreviously(Brillietal.,2010;Collier2012;Mohapatra,FioravantiandBiondi2014)
describingtranscriptionalregulators(DnaA,GcrA,CtrAandCcrM)actingsequentiallyandinisolation.Redlinesrepresenttranscriptional(activationorrepression)
controlsbytheregulators.OrangelinesrepresentmethylationcontrolbyCcrMonpromoters.Theasterisk(∗)representsthemethylationsite(5(cid:4)-GANTC-3(cid:4)).Thegreen
linerepresentsthephosphorylationofCtrA(CtrA-P)bytheCckAphosphorelay.Brokenlinescorrespondtoconnectionsthatremaincontroversialandquestionmarks
indicateelementsofthecircuitthatarenotunderstood.B.Newthree-tieredmodularnetworkshowingeachmodule:S-phase(boxedinblue)controlledbyGcrAand
CcrM,G2-phase(boxedinred)instatedbyCtrAandSciP,andG1-phase(green)dictatedbyCtrAandMucR.Theschemebelowthepanelshowsthecorresponding
timeinthecellcyclewhenthetargetpromotersofeachmodulefire.Thefluctuationoftranscriptsfromthesetargetgenesduringthecellcycleasdeterminedby
microarray(Laubetal.,2000;McGrathetal.,2007;Fangetal.,2013)andRNA-SeqanalysisisshowninFig.3.DnaAcontrolatthepromoterofthegcrA,podJandftsZ
genesisdepictedasdottedlinesbecausetheoccupancyofthispromoterbyDnaAhasonlydemonstratedinvitro,butnotyetbeenobservedinvivo.
Panisetal. 123
Rhodospirillales (Mercer et al., 2010; Bird and MacKrell 2011;
Greene et al., 2012; Zan et al., 2013; Wang et al., 2014) and
(iii) that these regulators bind a vast number of developmen-
tal promoters and regulatory sites in vivo (CtrA targets ca.
283 sites, GcrA ca. 218, SciP ca. 51 and MucR1/2 ca. 113/134)
(Fioravanti et al., 2013; Fumeaux et al., 2014). For the essen-
tial replication initiator DnaA, also classified previously as a
master transcriptional regulator (McAdams and Shapiro 2011;
Collier 2012), firm conclusions on its direct role in cell cycle
transcription (as opposed to indirect effects stemming from
itsessentialroleinreplicationinitiation),mustawaitmolecu-
lar or genetic dissection of the two activities of DnaA and/or
the determination of DnaA-bound promoters in vivo. On the
basis of this limitation, we put the contribution of DnaA
provisionally aside in this review, although we acknowledge
invitroexperimentssupportingthenotionthatDnaAactscon-
currentlywithorbeforetheGcrA/CcrMmodule(Hottes,Shapiro
andMcAdams2005).
TheGcrA/CcrMmoduleactivatesS-phasepromoters
TheGcrA/CcrMmoduleisthefirstepigeneticregulatorypairre-
portedtohaveaglobalroleincellcycletranscriptioninbacte-
ria.PhylogeneticanalysisrevealsthatcodingsequencesforGcrA
andCcrMgenerallyco-occurinalpha-proteobacterialgenomes
(Brillietal.,2010;Murrayetal.,2013)(Fig.1B).GcrAisinduced
withtheonsetofDNAreplicationinC.crescentuscells(Hottes,
Figure3.GcrA/CcrM,CtrA/SciPandCtrA/MucRtranscriptionalmodulesrespec-
ShapiroandMcAdams2005;Collier,MurrayandShapiro2006)
tivelycontrolsequentialwavesoftranscriptioninS-,G2-andG1-phase.For
and binds S-phase promoters in vivo (Fioravanti et al., 2013;
eachmodule,alistofvalidatedtargetgeneswasgeneratedbasedontheChIP-
seq data from Holtzendorff et al. (2004), Gora et al. (2010), Tan et al. (2010), Murrayetal.,2013).MostofthepreferredtargetsofGcrAharbor
Fioravantietal.(2013),Murrayetal.(2013),Fiebigetal.(2014)andFumeaux m6A GANTC marks introduced by CcrM (Zweiger, Marczynski
etal.,2014).Prerequisitestobeselectare:(i)expressionofthetargetgeneisde- andShapiro1994)(Fig.4).However,asCcrMonlyaccumulates
pendentofregulatorsoftheselectedmodule(publishedmicroarraysorpromoter after these S-phase promoters fire (i.e. in G2-phase), GANTC
reporterassays,seecitationsabove)and(ii)thisregulationmustbedirectasthe
methylationmustoccurintheG2-phaseofthepreviouscellcy-
promoterregionofthetargetgeneisbind(atleast)bytheconsideredregulators
cle,facilitatingtherecruitmentofGcrAtothesepromotersin
ofthemodule(publishedChIP,ChIP-seq,footprintingorEMSAexperiments).For
eachtranscriptionalmodule,anaveragevalueofthetemporalexpressionpro- theS-phaseoftheensuingcellcycle(Fioravantietal.,2013).
file[fromMcGrathetal.(2007)]oflistedgeneswascalculatedandnormalized GANTC methylation clearly enhances GcrA binding to its
tomaximalexpression.Thisaveragetemporalexpressionprofileisdepictedas target promoter in vitro and in vivo (Fioravanti et al., 2013). In
sequentialwavesofactivitiesofGcrA/CcrM,CtrA/SciPandCtrA/MucRtranscrip- vitro GcrA binds its target promoters even in the absence of
tionalmodules.
methylation, but the binding is enhanced in vivo and in vitro
whentheoverlappingGANTCmotif(s)carriesanm6Amethyla-
eitherdirectlyand/orindirectly(Quonetal.,1998;Bastedoand tionmark(Fioravantietal.,2013).Suchmethylation-dependent
Marczynski 2009), binds and induces the accumulation of G2- binding in vitro has also been observed for GcrA from other
andG1-phase-specifictranscripts.CtrAactswithdifferentneg- alpha-Proteobacteria(Fioravantietal.,2013),suggestingthatthis
ativeregulatorstorestrictpromoterfiringtothecorrectcellcy- mechanismofGcrA-dependentpromoterrecruitmentandpre-
cle phase (Laub et al., 2000, 2002; Fiebig et al., 2014; Fumeaux sumably activation is conserved in many alpha-Protebacteria.
et al., 2014). The G2-phase transcriptional module comprises However, not all GcrA target promoters harbor GANTC se-
CtrAandthehelix-turn-helixdomainproteinSciPthatisonly quences(Fioravantietal.,2013;Murrayetal.,2013).Conversely,
presentinG1-phase(Goraetal.,2010;Tanetal.,2010)andtargets GANTCmethylationbyCcrMisclearlynotsufficientforthere-
theG2-classofCtrA-activatedpromoters(Fumeauxetal.,2014) cruitmentofGcrAtoitstargets,asindicatedbythefactthatGcrA
(Fig.3).Bycontrast,theMucRparalogs(henceforthMucR1/2)re- has a clear preference for certain sites carrying m6A GANTC
sembleancestralZinc-fingerproteins(Close,TaitandKado1985; marks in vivo, but several other GANTC sites are also methy-
Malgierietal.,2007;Baglivoetal.,2009)andbindthepromotersof lated and are not efficiently bound by GcrA in vivo (Fioravanti
G1-phasegenes(Fumeauxetal.,2014).Thus,MucR1/2andCtrA etal.,2013;Murrayetal.,2013).Thus,additionaldeterminants
controltheG1-phasetranscriptionalmodule. orpossiblyothermethylases(Kozdonetal.,2013)mustalsocon-
GcrA,CcrM,SciPandCtrAhaveeachbeendescribedases- tributetotherecruitmentofGcrAtoitstargetsinvivo.
sentialmastercellcycleregulatorsinrecentreviews(McAdams ThemethylationstateofGANTCsiteschangesequentiallyas
andShapiro2011;Collier2012).Here,weinsteadrefertothese theDNAreplicationmachineryprogressivelycopiesthechromo-
cell cycle protein as ‘global regulators’ on the grounds of re- someandindoingsoconvertsfullymethylatedGANTCtothe
cent genetic experiments showing (i) that GcrA, CcrM, SciP hemi-methylated form (before CcrM is present, Fig. 4A). Thus
or MucR1/2 are dispensable in C. crescentus (Gora et al., 2010; uponreplication,thesegenomicGANTCsitesresideinahemi-
Gonzalez and Collier 2013; Murray et al., 2013; Fumeaux methylatedstateuntilre-methylatedbyCcrMonceitaccumu-
et al., 2014), (ii) that CtrA can be inactivated in several alpha- latesinG2-phase.Bycontrast,Cori-distalsites(neartherepli-
proteobacterial lineages including the Rhodobacterales and the cation terminus) remain fully methylated for most of the cell
124 FEMSMicrobiologyReviews,2015,Vol.39,No.1
Figure5.ComplexregulationoftheC.crescentusctrApromoterbymultiplecell
cycletranscriptionalregulators.A.Theschematicshowsthespatio-temporal
changesofCtrAduringthecellcycle.B.Thegraphshowsthefluctuationofthe
ctrAtranscriptduringthecellcycle,computedfromtheglobaltranscriptional
profilingdata(McGrathetal.,2007)normalizedtomaximalexpressionforeach
gene.C.ChIP-Seqtracesshowingtheoccupancyofvarioustranscriptionalreg-
ulatorsatthectrApromoterbasedondatafromFioravantietal.(2013),Murray
etal.(2013),Fiebigetal.(2014)andFumeauxetal.(2014).D.Regulationofthetwo
promotersofthectrAgene,P1andP2.TheweakerP1promoterfiresfirstandis
activatedbyGcrA,repressedbySciPandCtrA,andsilencedbyfullDNAmethy-
Figure4.TargetgenesoftheGcrA/CcrMtranscriptionalmoduleandtheirposi-
lation(∗representsmethylationsite).ThestrongerP2promoterisactivatedby
tionontheC.crescentusgenome.A.Replicationcontrolandtemporallyregulated
(re-)methylationoftheC.crescentuschromosomeat5(cid:4)-GANTC-3(cid:4) sitesduring CtrAinapositivefeedbackloop.MucR1/2bindtothectrApromoterandwere
alsorecentlyshowntoinfluencectrAexpression(Fumeauxetal.,2014).Thisin-
cellcycleprogression.Caulobactercrescentushasa4.01Mbpchromosomethat
terconnectedregulationallowsprogressivectrAexpressionthroughoutS-and
isreplicatedonlyonceduringthecellcycleandatdifferenttimesinthedaugh-
G2-phase(seeFig.5A).ThectrApromoterisalsoboundbytheStaRtranscription
tercellsafterdivision.ThisreplicativeasymmetryisnegativelycontrolledinG1
byCtrA.FollowingthedegradationofCtrAduringtheG1→Stransition,DnaA factorasdescribedbyFiebigetal.(2014).
assemblesonCoritopromotetheinitiationofchromosomereplication.CtrA
re-accumulatesduringthemiddleofS-phaseandpreventsre-initiation(multi- cycle as they are replicated only later (Fig. 4B). Such gradual
forkreplication).Concurrently,withprogressionofthereplicationforkduring changeinmethylationstateduringthereplicationcycleoffers
S-phase,theGANTCsequencesareconvertedfromafull-methylatedstateto
asimpleandappealingwaybywhichcellscancoordinatetran-
ahemi-methylatedstate.DNAre-methylationonlyoccursinG2-phaseonce
scription of selected genes with the cell cycle (also known as
theCcrM-specificmethylase(methylatesGANTCattheN6-positionofadenine)
accumulates.B.ManyvalidatedtargetgenesoftheGcrA/CcrMtranscriptional the‘methylationratchet’model)(Collier,McAdamsandShapiro
modulelocalizeneartheterregion.Aputativegenelistdirectlycontrolledby 2007).Suchamechanismmaywelldictatethetimingoftran-
theGcrA/CcrMmodulewasgeneratedbycombiningthegeneexpressiondata scriptionofthectrAgeneduringthecellcycle(Domian,Reise-
ofGcrA-depletedcells(Holtzendorffetal.,2004),GcrApromoteroccupancyas nauer and Shapiro 1999). The ctrA gene is located at a proxi-
determinedbyChIP-seqanalysis(Fioravantietal.,2013;Murrayetal.,2013)and
mal position to the C. crescentus origin of replication (Cori) on
m6A-methylationdataasdeterminedbyChIP-seqanalysisandglobalmethy-
the circular chromosome and the ctrA P1 promoter fires early
lomeanalysis(Fioravantietal.,2013;Kozdonetal.,2013).Inagreementwithpub-
lisheddataonGcrA/CcrMdependenceusingtranscriptionalreporterstrains(Fio- in S-phase, coincident with the passage of the DNA replica-
ravantietal.,2013;Murrayetal.,2013),tipF,ftsN,podJ,mipZandpleCgeneswere tionforkpastthectrAlocus.Bycontrast,mostofthepreferred
retainedinthisselectionof37putativecontrolledgenes.Unfortunately,ctrAand GcrAtargetpromotersresideatCori-distalpositions(Nierman
ftsZarenotincludedinthelistastheywerealreadyabsentintheGcrA-regulated etal.,2001)(Fig.4A–C),yetthetranscriptsofthesegenesarein-
geneslistfromHoltzendorffetal.(2004).Thepositionoftheselectedgenesasa
duced concurrently with ctrA (Laub et al., 2000). The fact that
functionoftheirlocationontheC.crescentuschromosomeisdepicted.C.Sum-
GcrA-dependentpromotersatbothofthesechromosomalloca-
maryofallknowninteractionsofthetranscriptionalregulatorsatthepromoters
ofvalidatedgenescontrolledbytheGcrA/CcrMtranscriptionalmodule.Aster- tionsfireinS-phaseimpliesthatthetimingofpromoteractiva-
isks(∗)representthemethylationsite(5(cid:4)-GANTC-3(cid:4))inthedifferentpromoters. tionbyGcrAisnotdirectlycoupledtothechangeinmethylation
DnaAcontrolatthepodJandftsZpromotersisdepictedasdottedarrowbecause stateofthepromoterinducedbypassageoftheDNAreplication
theoccupancyatthispromoterbyDnaAinteractionhasonlydemonstratedin forkpastthecorrespondinglocus(i.e.notcorrelatedwiththe
vitro(Hottesetal.,2005),butnotyetbeenobservedinvivo.
changeinmethylationstatefromfull-tohemi-methylation).It
thereforeseemsthatthemodelthatthereplicationforkpassage
istiedtothetemporalactivationofpromotersisnotageneral
feature for GcrA/CcrM-regulated S-phase promoters. One pos-
sibleexceptiontothiscouldtobetheP1promoterofthectrA
gene(henceforthctrAP1,Fig.5D)andperhapsthepromoterof
Panisetal. 125
thelongnon-codingRNACCNAR0116,originallyannotatedas theGcrA/CcrMregulatorypairtogethermayservetocoordinate
hypotheticalgeneCCNA00697(Marksetal.,2010).Unlikemost fastandefficientgrowthanddivisioninthefree-livingalpha-
otherGcrA-dependentpromoters,mutationofGANTCsitesin proteobacteriallineages(Fig.1B).
these two promoters does not cripple promoter activity, pro- Unexpectedly,jointinactivationofgcrAandccrMinC.cres-
videdthatthemutationitselfdoesnotinadvertentlyincrease centusdidnotaccentuatethedivisiondefectofthesinglemu-
theaffinityofRNApolymerase(RNAP)forthemutantpromoter tantsandrenderthemnon-viable,butinsteadhadtheopposite
(Reisenauer and Shapiro 2002; Fioravanti et al., 2013). By con- effect:itimprovedgrowthandelevatedsteady-statelevelsofsev-
trast, ctrAP1 is less active when it is moved from its normal eraldivisionproteinscomparedtothesinglemutants(Murray
Cori-proximalpositiontoaCori-distalsiteonthechromosome etal.,2013).ThisindicatesthatinisolationGcrAorCcrMaffect
(ReisenauerandShapiro2002),suggestingthatforthispromoter cellcycleprogressioninanadversewayintheabsenceofthe
theremightindeedbeacorrelationbetweenctrAP1activityand otherandthatitismorebeneficialforcellstohavebothorto
the time it resides in the hemi-methylated state. GcrA bind- lack both, rather than having only one of the two. This trend
ingtothispromoterisenhancedbyGANTCmethylationinvitro isrecapitulatedbythephylogeneticanalysisshowninFig.1B
andinvivo(Fioravantietal.,2013).However,GcrAexhibitedthe (withthecaveatthatmethylasesofrestrictionmodificationsys-
highestaffinityforthefullymethylatedctrAP1promoterver- tems can be erroneously annotation as CcrM orthologs). The
sus the hemi-methylated promoter (and more affinity for the antagonisticgeneticrelationship(wheregrowthdefectsfroma
hemi-methylated than the non-methylated promoter) in vitro loss-of-functionmutationinonegenecanbepartiallycompen-
(Fioravantietal.,2013).Theseresultsmaybereconciledwiththe satedbyaloss-of-functionmutationinthesecondgene),along
notionthatGcrAcanacteitherpositivelyornegativelyonthe withtheaforementionedbiochemicaldependencies,couldex-
ctrApromoterasafunctionofthemethylationstate,resulting plainthelossofgcrAandccrMintheobligateintracellularalpha-
inanapparentlyneutraleffectonpromoteractivityintheab- Proteobacteriaortheiracquisitionbythefree-livinglineagesto
senceofmethylation.Twolinesofevidencesupportthisnotion. permitrapidadvancementthroughtheS-phaseprogramwhen
First,invitrofootprintingrevealedthedifferentextentofbind- needed.
ing at methylated versus hemi-methylated ctrAP1 (Fioravanti WhatisthemolecularbasisfortheadverseeffectsofGcrA
etal.,2013),withtheformeryieldingalargerprotectionthatcov- on growth and division when CcrM is absent and vice versa?
erstheentirepromoter,whilethelatterisconfinedtothe−35 SinceGcrAcaninteractwithRNAPandcancoveralargearea
siteandthejuxtaposedGANTCsite.Second,mutationalanalysis ofthepromoterinamethylation-dependentmanner(Fioravanti
inGcrAdepletionstrainssuggestthatGcrAactsnegativelyinthe etal.,2013),itisconceivablethatGcrAplaysaroleintheproper
absenceofmethylation(oftheftsNpromoter,seebelow),while positioningofRNAPatthepromoterforactivationor,whenit
itactspositivelywhenthesiteismethylated(Murrayetal.,2013). isimproperlyregulated,interfereswithtranscriptionalactiva-
tion,forexamplebytetheringRNAPtothepromoterandpre-
ventingclearanceofRNAPonafullymethylatedpromoter.In-
LifeanddivisionwithoutGcrAand/orCcrM
deed,depletionofGcrAcripplesthewild-typeftsNpromoter,but
ThetargetpromotersofGcrA/CcrMinC.crescentusincludethe causesanincreaseinactivityoftheftsN‘GANTCmutant’pro-
promotersof dispensablegenesencoding polarityfactors and moter,likelyduetode-repression(Murrayetal.,2013)(Fig.4C).
regulators(e.g.podJ,pleC,tipFandflaEY;Figs2Band4BandC)that WhilepromotermethylationbyCcrMappearstoconvertGcrA
promoteflagellumassemblyandtheextrusionofpiliatthenew from a negative regulator of transcription into an activator, it
pole(oppositethestalk)(Davisetal.,2013;Fioravantietal.,2013; is unclear why CcrM acts negatively in the absence of GcrA.
Murray et al., 2013). GcrA/CcrM also regulates a number of It is possible that CcrM performs functions independently of
other target promoters that promote expression of essential methylationorthatmethylationservesacriticalbutunknown
celldivisiongenes,suchasftsNandftsZ(MollandThanbichler roleinalpha-proteobacterialcellphysiologyotherthancellcy-
2009;GonzalezandCollier2013;Murrayetal.,2013),indicating cletranscription(Gonzalezetal.,2014).Alternatively,themere
that GcrA/CcrM controls other essential functions in addition presence(binding)ofCcrMatthepromoterduringmethylation
toCtrA. oftheGANTCmotifmightsimplyinterferewithresidualtran-
Interestingly,theGcrA/CcrMmodulewasrecentlyshownto scriptionfromacrippledpromoterwhenGcrAisabsent(Murray
bedispensableinseveralalpha-Proteobacteria(Fig.1B),atleast etal.,2013).Inthiscase,eliminationofsuchaninterferingac-
when cells grow slowly (Murray et al., 2013; Curtis and Brun tivityontranscriptionwithaccrMmutationcouldexplainthe
2014).ThefactthatnoorthologsofGcrAandCcrMareencoded fairlymodest(2-fold)increaseinthesteady-statelevelsofthe
inthesequencedgenomesofWolbachiaandRickettsiasp.(Brilli cell division proteins FtsN and MipZ seen in the C. crescentus
etal.,2010)indicatesthatGcrA/CcrMisnotrequiredforsurvival gcrA/ccrMdoublemutantversusthegcrAsinglemutant(Murray
withineukaryotichostcells,possiblyreflectinggrowthatare- etal.,2013).Onthescaleofanindividualcelldivisionprotein,
ducedrate.DisruptionofeithergcrAorccrMinBrevundimonas theincreaseinabundanceincelldivisionproteinssuchasFtsN
subvibrioides,aslowgrowingspeciesfeaturingadoublingtime orMipZisquitesmall;however,thecumulativesmallincrease
4-foldlongerthanthatofwild-typeC.crescentus,isapparently in all GcrA/CcrM-regulated cell divisions proteins could easily
possiblewithoutadverseeffects(CurtisandBrun2014).More- amounttoastrongbeneficialeffect,explainingwhytheselec-
over,C.crescentuscellslackingeitherCcrMorGcrAgrowslowly tivepressurewassufficientlystrongtounearthaccrMtranspo-
andareveryfilamentousowingtoashortageindivisionpro- son(ccrM::Tn)mutationthatimprovesgrowthofGcrA-deficient
teins(GonzalezandCollier2013;Murrayetal.,2013).Itiscon- C.crescentuscells.Ontheotherhand,experimentsshowthatel-
ceivablethatwhengrowingslowlythesemutantshavesufficient evatedexpressionofFtsNaloneissufficienttoamelioratethe
timetoaccumulateenoughdivisionproteinsrequiredtoassem- growthdefectsofgcrAorccrMmutantcells(Murrayetal.,2013).
bleacompletecircumferentialcytokineticstructuretodivide(de Itisknownthatoverexpressionofcriticallatedivisioncompo-
Boer2010).Bycontrast,celldivisionseemsinsupportableunder nents such as FtsN can overcome shortages in other division
fastgrowthresultingindeathduetoafailuretodivide.Thus, proteins in Escherichia coli simply because overexpression can
126 FEMSMicrobiologyReviews,2015,Vol.39,No.1
stabilize the division machine or can confer sufficient critical Whydifferentbacteriarelyonmethylaseswithdifferentki-
cellwallbiosyntheticactivitytopromoteconstriction(Bernard neticpropertiesformethylationcontrolisaninterestingques-
etal.,2007;Gerdingetal.,2009). tionnotpreviouslydiscussed.Owingtothedistributivemodeof
action,thedwelltimeofCcrMonDNAislikelyshortandmay
MolecularbasisofphenotypicdifferencesbetweengcrA limit target interactions. As CcrM is a dimeric enzyme (Shier,
HanceyandBenkovic2001)andmonomericCcrMcanbindand
andccrMmutants
methylate DNA, dimerization could endow CcrM with the ca-
DespitethemanycommonalitiesbetweenGcrAandCcrM,there pacitytomethylatetwohemi-methylatedtargetsitessimulta-
arealsoimportantphenotypicdifferencesinthemutantstrains neously(oneperCcrMmonomer,forexamplejuxtaposedsites
that provide valuable clues about the regulation of key target on the same chromosome or the same loci on the two sister
genes. For example, ccrM-deleted cells are elongated without chromatids),concomitantlyincreasingitsdwelltimeonthesu-
obviousconstrictions,whilegcrA-deletedcellsareimpairedat percoiled DNA in vivo at promoters with different numbers of
alatestageofdivisionandshowconstrictions.Thisdifference GANTCsites.Despitea5-foldhighernumberofDammethyla-
pointedtoadefectinFtsZaccumulationintheformer,butnotin tionsites(ca.20000)thanCcrM(4542)inC.crescentus,thecel-
thelatter(Murrayetal.,2013;Gonzalezetal.,2014).WhileDnaA lularconcentrationofDaminE.coliismorethan23-foldlower
isknowntobindtheftsZpromoter(andotherssuchasthepodJ thanthatofCcrM(ca.130moleculesversus3000moleculesper
promoter)invitro(Hottes,ShapiroandMcAdams2005),itispos- cell,respectively)(Shier,HanceyandBenkovic2001)andmaybe
siblethatCcrM-dependentcontrolofDnaAattheftsZpromoter evenfurtheraccentuatedbythemulti-forkreplicationinE.coli
underliesthedifferencesinthecelldivisionphenotypebetween comparedtothenon-overlappingoneinC.crescentus.Proteolytic
thegcrAandtheccrMmutants. degradationofadistributiveenzymemightensurearapiddrop
Alternatively,differencesinCtrAlevelscouldaccountforthe insteady-statelevelsbelowthethresholdneededformethyla-
lowerlevelsofFtsZaccumulationintheccrM::Tnmutantcom- tion,whilealownumberofprocessivemethylasemoleculesthat
paredtothe(cid:2)gcrAor(cid:2)gcrAccrM::Tnstrains(Murrayetal.,2013; escapedegradationorarisefromstochasticsynthesismightsuf-
Gonzalez et al., 2014). CtrA directly represses the ftsZ (and ficetokeepthegenomefullymethylatedatalltimes.
podJ)promoter(Kellyetal.,1998;Crymes,ZhangandEly1999; InC.crescentus,CcrMdegradationdoesnotseemtooccurat
Fiebig et al., 2014; Fumeaux et al., 2014) (Fig. 4C). While (cid:2)gcrA aspecifictimeinthecellcycle(Wrightetal.,1996),butaburst
mutants exhibitreduced CtrA abundance, ccrM::Tnand(cid:2)gcrA inccrMtranscriptioninG2-phasedictateswhentheenzymeac-
ccrM::Tn mutants accumulate CtrA to near wild-type levels cumulates(Stephens,ZweigerandShapiro1995)(Fig.6C).InS.
(Murray et al., 2013), suggesting that ftsZ is more strongly re- meliloti,theccrMtranscriptalsopeaksatthelatepre-divisional
pressedinthegcrA/ccrMdoublemutantcomparedtothe(cid:2)gcrA (G2)cellstage(DeNiscoetal.,2014)andinAgrobacteriumtume-
singlemutant(Murrayetal.,2013).ThectrAtranscriptlevelsare faciens re-methylation commences at or near completion of
at best slightly upregulated in the absence of CcrM (Gonzalez DNAreplication(KahngandShapiro2001),correspondingtothe
etal.,2014),consistentwiththenotionthatDNAmethylation times when CcrM is expressed and active in C. crescentus and
doesnotaffectctrApromoteractivityquantitatively(seeabove), S.meliloti.Nevertheless,themerepresenceofCcrMinthecell
incontrasttotheknowndown-regulationwhenGcrAisdepleted doesnotguaranteethatagivenGANTCsiteisindeedmethy-
(Holtzendorffetal.,2004).Insupportofthis,thedown-regulation lated.Recentstate-of-the-artmethylomeanalysesatsinglebp
ofctrAP1inthegcrAmutantresemblesthatofstraininwhich resolutioncapturedtheprogressivehemi-methylationandsub-
ctrAP1wasinactivatedbyafivebasepair(bp)insertionbetween sequentre-methylationof4515GANTCsitesatfivetimepoints
the−35and−10sites,inthemiddleoftheGcrA-bindingregion duringtheC.crescentuscellcycle(Kozdonetal.,2013).Interest-
(Schredletal.,2012).IfthereductioninCtrAissolelyduetothe ingly,27sitesremainedunmethylatedthroughoutthecellcycle.
inabilityofmutantctrAP1tobeactivatedbyGcrA,thenCcrM Three of these sites are located in intergenic regions that are
shouldalsostillactnegativelyonthismutantpromoterakinto notpermissiveforintegrationoftheTn5transposon(Christen
thewild-typepromoteringcrAmutantcells,assuggestedbythe etal.,2011).Astheseregionscanbedeleted(Kozdonetal.,2013),
fact thatinactivationofccrMingcrAcellsrestoresCtrAlevels theyareclearlydispensableforviability,suggestingthattheyare
(Murrayetal.,2013).Indeed,theGANTCremainsintactinthis notaccessibletoTn5-mediatedinsertionforotherreasons.As
ctrAP1mutant(Schredletal.,2012),suggestingthatGcrAbind- constitutiveexpressionofCcrMthroughoutthecellcyclefrom
ingisdisturbedatthemutantpromoterandthatCcrMcanstill aconstitutivepromoterresultedinmethylationofthesesites
actnegativelyonthismutantpromoter. (Gonzalezetal.,2014),transpositioncouldbetemporallyregu-
latedorthesitesareprotectedfrommethylationonlyatspe-
cifictimesduringthecellcycle.Itisconceivablethatchromatin
CcrMisadistributive,notaprocessive,DNAmethylase
topology in G2-phase masks this region or that a G2-specific
ThenegativeroleofCcrMintheabsenceofGcrAcallsforfur- DNA-bindingproteinoccludestheseunmethylatedsites.Thus,
therstructure-functionandbiochemicalstudiesofCcrM,espe- specificmechanismsexistthatmayrestrictepigeneticmodifi-
cially in light of the unusual ca. 80-residue C-terminal exten- cation. Interestingly, several of these unmethylated sites also
sion of unknown function that is present only in CcrM-type overlap with known binding sites for MucR in C. crescentus
alpha-proteobacterial methylases. It has been proposed that (Fumeauxetal.,2014),raisingthepossibilitythatMucRprotects
CcrM might depend on an accessory factor that helps to load from methylation by CcrM. It is currently unknown if GANTC
themethylaseontotheDNA(Albu,JurkowskiandJeltsch2012), sites in other alpha-Proteobacteria show similar methylation
asthebiochemicalmechanismofactionofCcrMisverydistinct protection.
fromthatofDamfromE.coliandcoliphageT4.WhileC.cres-
centusCcrMisnowknowntobedistributiveenzymeinvitro,the
ControlofG2-phasepromotersbytheCtrA/SciPmodule
aforementionedDamenzymesfeatureaprocessivemodeofac-
tion(Berdisetal.,1998;Zinovievetal.,2003;PetersonandReich With the activation of ctrA transcription in S-phase by the
2006;Albu,JurkowskiandJeltsch2012). CcrM/GcrA circuit, the stage is set for next transcriptional
Panisetal. 127
Figure6.AbundanceofGcrA,CcrM,SciPandMucRandtheirmRNAsduringtheC.crescentuscellcycle.A–D.Theschematicontheleftshowsthespatio-temporal
changesofeachregulatorduringthecellcycle.Thegraphsinthemiddleshowthefluctuationofthetranscriptduringthecellcycle,computedfromtheglobal
transcriptionalprofilingdata(McGrathetal.,2007)normalizedtomaximalexpressionforeachgene.Theschematicontherightsummarizesallknowninteractions
ofthetranscriptionalregulatorsatthepromotersoftherespectiveregulatorgenes.A.SciPaccumulationduringtheG1-phase.SciPispresentinG1-phaseand
protoelyticallyremovedduringtheG1→Stransition.Re-synthesisofCtrA(inlateS-andearlyG2-phases)precedesthatofSciP(inG1-phase).B.GcrAaccumulation
duringS-phase.DnaAcontrolatthepromoterofthegcrAgeneisdepictedasdottedarrowbecausetheoccupancyofthegcrApromoterbyDnaAinteractionhasonly
demonstratedinvitro,butnotyetbeenobservedinvivo.C.FluctuationofCcrMandregulationoftheccrMpromoter.Theasterisk(∗)representsthemethylationsite
(5(cid:4)-GANTC-3(cid:4))intheccrMpromoter.D.MucR1,MucR2andDnaAaregroupedtogetherasthesethreeregulatorsareexpressedatdifferenttimeduringthecellcycle
buttheirsteady-statelevelsdonotfluctuatesignificantly.
moduletoact:theCtrA/SciPmodulethatactsinG2-phase.Upon competition assays revealed that CtrA recruits RNAP to these
itssynthesis,CtrAisphosphorylated(CtrA∼P,Fig.2and5A-B, promoters(Goraetal.,2010).Suchrecruitmentisperturbedin
buthenceforthjustreferredtoasCtrA)andactivatesexpression thepresenceofSciP,presumablybecauseSciPandRNAPcom-
offlagellarstructuralproteins,chemotaxisproteinsandother peteforthesamebindingsurfaceinCtrA.Interestingly,these
developmental factors in the G2-phase of C. crescentus (Do- findings point to a mechanism by which SciP silences its tar-
mian, Quon and Shapiro 1997; Laub, Shapiro and McAdams getpromotersinvivowithoutdisplacingCtrAfromthepromoter
2007) (Fig. 3), while repressing other genes such as ftsZ, podJ (Gora et al., 2010). As the DNA·CtrA·SciP ternary complex was
(Laub et al., 2000; Fiebig et al., 2014) and gcrA (Holtzendorff showntoprotectCtrAandSciPfromtheproteasesClpXPand
et al., 2004). CtrA activates the G2-specific genes until SciP Lon,respectively,stabilizationofbothproteinscanaffectcellcy-
represses them upon entry into G1-phase (Chen et al., 2006; cleprogression(Goraetal.,2013).Evidently,theinteractionbe-
Fumeauxetal.,2014)(Fig.6A).ThisisbecausethesciPgene,en- tween SciPandCtrAisnotonlyanimportant determinant of
codingtherepressor/negativeregulatorofG2-phasepromoters, CtrAproteolysis,butiscriticalforSciPfunction,asindicatedby
isitselfpartoftheG1-phasegenesthatCtrAactivatesduring thefindingthatwhenSciP’sabilitytobindCtrAiscompromised,
cytokinesis(Fumeauxetal.,2014).Aconserved93-residuepro- itcannolongerefficientlyinhibittranscriptionofCtrA-activated
teinwithawingedhelix-turn-helixDNA-bindingdomain,SciP, genes(Goraetal.,2010;Tanetal.,2010).
isproteolyzedduringtheG1→StransitionbytheLonprotease ThefindingthatSciPdoesnotefficientlyassociatewiththe
(Goraetal.,2010,2013;Tanetal.,2010)(Fig.6A).InvivoSciPhasa promoters of G1-phase genes in vivo suggests that this CtrA-
strongpreferenceforpromotersthatCtrAactivatesinG2-phase dependent interaction is weak or very short-lived (Fumeaux
versusthepromotersofG1-phasegenes(Fumeauxetal.,2014). etal.,2014).Bycontrast,SciPefficientlyassociateswithpromot-
InvitroSciPhasonlyveryweakorbarelydetectablegeneralDNA- ersthatareactivatedbyCtrAinG2-phase.UsingthesetopSciP
bindingabilityonCtrAtargetpromotersthatarenotamongits invivotargetsites,aconsensusmotifforSciPtargetDNAhas
preferredinvivotargets,butCtrAdirectlyboundtothesepro- beenproposed[5(cid:4)-(G/A)TTAACCAT(A/G)-3(cid:4)](Fumeauxetal.,2014)
moterscanrecruitSciP(Goraetal.,2010,2013).Moreover,invitro that is remarkably similar to the ‘extended’ CtrA target motif
128 FEMSMicrobiologyReviews,2015,Vol.39,No.1
[5(cid:4)-TTAACCAT-3(cid:4)](Spenceretal.,2009).Thisextendedmotifisa belargelyduetocontrolofproteinsynthesis,asevidencedby
variationofthe‘standard’orclassicalCtrArecognitionmotif[5(cid:4)- driving its expression from the xylose-inducible promoter for
TTAA-N7-TTAA-3(cid:4)]andisboundinvitrounderconditionsthat constitutive induction during the cell cycle (Gora et al., 2013).
artificiallyfavorthedimerizationofC.crescentusCtrA(Spencer RegulationofSciPsynthesisnormallyrestrictsitsaccumulation
etal.,2009).MightthereforeSciPpromotedimerizationofCtrA toG1-phase(Fig.6A).InC.crescentus,theparalogousrepressors
ordepend onit? Thefact findingthat regionsthat SciP binds MucR control transcription of G1-specific genes that are acti-
in vivo are slightly juxtaposed but not completely overlapping vated by CtrA such as sciP (Fumeaux et al., 2014) (Fig. 2B and
withtheCtrApeakssuggeststhatSciPcouldassociateperiph- 3).AsMucRdonottargetCtrA-activatedpromotersthatfirein
erallyCtrAontargetpromotersatsuch‘extended’CtrAmotifs G2-phase,asimplemodelwasadvancedinwhichde-repression
(Fumeauxetal.,2014).Adifferentconsensusmotifwiththecore ofG1-phasegenesdirectstheaccumulationofSciP,whichwill
GC-richsequence5(cid:4)-GTCGC-3(cid:4)wasadvancedbyTanetal.(2010). leadtoanimmediateshutdownofG2-phaseCtrA-activatedpro-
Whiletheformermotifwasproposedbasedexclusivelyonthe moters(Fumeauxetal.,2014).TheG1-specifictranscriptlevels
50preferredinvivotargetsitesof(untagged)SciPasdetermined couldriseuntilathresholdofSciPisreachedthatwoulddis-
byChIP-Seqexperiments(Fumeauxetal.,2014),withoutfurther lodgeRNAPfromallCtrA-activatedpromoters(Goraetal.,2010)
experimental verificationsuchasmutagenesis,thelattermo- oruntilCtrA/SciPisdegradedduringtheG1→Stransition(Do-
tifwasderivedonweakandconcentration-independentprotec- mian,QuonandShapiro1997;Goraetal.,2013).Inthismodel,
tioninfootprintingstudiesusingHis6-SciP(SciPharboringanN- MucRadoptsacriticalroleinregulatingG1-specificgeneexpres-
terminalahexahistidinetag)andthectrApromoter,arelatively sionand,byextension,mayactasthetriggerfortheswitchfrom
poor in vivo substrate of SciP (Fumeaux et al., 2014), and rein- theG2-phasetranscriptionalprogramtotheG1-phaseprogram
forcedwithlower-resolutionChIPdataofFLAG-SciP(SciPhar- (Fumeauxetal.,2014).AsMucRsteady-statelevelsdonotfluc-
boringanN-terminalFLAGtag)andindirecttranscriptprofiling tuate significantly during the cell cycle (Fumeaux et al., 2014)
data.Thereasonfordiscrepancyisnotclear,butmutagenesis (Fig.6D),themechanismofactivationisprobablyoccurringat
experimentsshowedthatmutationoftheproposed5(cid:4)-GTCGC- thelevelofMucRactivity.
3(cid:4) motifdoesnotalterpilApromoteractivity(Goraetal.,2013). MucR proteins feature an ancestral zinc finger-type DNA-
ThisconclusionisconsistentwiththefindingthatSciPdoesnot binding domain, comprising a recognition helix (α) and a
bindthepilApromoterinvivobutveryefficientlytargetsthepro- two-stranded beta-sheet (β) embedded within a central ca.
motersofmotilitygenesinvivo(Fumeauxetal.,2014).Withthe 58-residue globular domain of βββαα architecture (Malgieri
identificationofmotilitypromotersastopSciPinvivotargets, et al., 2007; Baglivo et al., 2009). How DNA binding of MucR
thestageisnowsettorevisitthebindingpropertiesandtarget and conserved alpha-proteobacterial orthologs might be reg-
sequenceinvitrobyfootprintanalysisusingthesepromotersas ulated is unclear, but the N- and C-termini could certainly
probes. provideentrypointsforpost-translationalregulation.Biochem-
ThoughSciPhasalsobeenimplicatedinregulationofmotil- icalstudieshaveshownthatDNA-bindingofS.melilotiandBru-
ityinRhodobactercapsulatus(Merceretal.,2012),C.crescentuscells cella abortus MucR depends strongly on the presence of diva-
lackingSciParenotonlynon-motile,butalsogrowslowlyand lentcations(Bertram-Drogatz etal.,1998;Caswelletal.,2013).
arefilamentous(Goraetal.,2010).Whytheabsenceofnegative InsupportoftheproposedimportantroleofMucRinswitch-
regulatoroftranscriptionhassuchadetrimentaleffectoncell ing transcriptional programs in C. crescentus, the mucR muta-
physiologyisnotintuitive,sincemanycellcycleproteinsarereg- tion is pleiotropic in several lineages where mucR has been
ulatedatthelevelofabundanceanditisunknownwhichgenes’ geneticallyanalyzed(CooleyandKado1991;Bittingeretal.,1997;
prematureexpressioncouldhavesuchdramaticconsequences. Martinetal.,2000;Bahlawaneetal.,2008;MuellerandGonzalez
Clearly,theexpressionofatleastoneSciPtargetgenemustbe 2011;Mirabellaetal.,2013).ManyMucRorthologsfromdiffer-
properlynegativelyregulatedtoavoidadverseeffectsongrowth entalpha-Proteobacteriaareinterchangeableandcansupport
and/ordivision.AsSciPdoesnotassociatewithtargetsthatCtrA MucRfunctioninC.crescentusorS.meliloti(Mirabellaetal.,2013).
negativelyregulates,suchasCoriandthepodJandftsZpromot- Moreover, pangenomic ChIP-seq analysis revealed that MucR
ers,invivo(Fumeauxetal.,2014)(Fig.4C),itseemsthatexcessive fromS.frediitargetsseveralpromotersofgenesorthologousto
firingofCtrA-activatedpromotersmustaccountforthepheno- thosethatMucR1/2targetsinC.crescentus(Fig.5C-D)(Fumeaux
typicproblemsofsciPmutants.Insupportofthis,CtrAtarget etal.,2014).ItisthereforelikelythattheroleofMucRincon-
sitesinCoriaredispensableforviability(BastedoandMarczyn- trollingcellcycletranscription(andbyinferenceitsregulation)
ski2009)andCtrAdoesalsonotappeartobindatorwithinthe isconservedinthislargeclassofbacteriathatoccupydistinct
originofreplicationofS.fredii(Fumeauxetal.,2014). ecological niches and that the basic architecture of the reg-
ulatory network has been maintained in these different lin-
eages (Fig. 1B). While ecological adaptation probably resulted
ControlofG1-phasegenesbytheCtrA/MucRmodule
in the recruitment of niche-specific functions into the MucR
ThesecondtranscriptionalmodulethatCtrAspecifiesinC.cres- regulon, there are compelling hints that MucR controls G1-
centusistheG1-phasemoduleinwhichCtrAactivatespromot- specificfunctionsinothersystems,specificallythosethatare
ersofG1-phasegenes,whileMucR1/2repressthempriortoen- requiredforinteractionswithhostcells.Forexample,theG1-
tryintoG1(Fumeauxetal.,2014).Whilethemechanism(s)by phasewasrecentlyshowntoharborthefunctionsrequiredfor
whichMucR1/2repressionislostorcanbeovercomeiscurrently hostcellinvasionbyB.abortus(Degheltetal.,2014).Moreover,a
unknown,MucR1/2clearlyadoptacriticalroleininstatingG1- B.melitensisoraB.abortusmucRmutantisattenuatedininfec-
phasegeneexpressionandturningoffG2-phasepromotersvia tionofmacrophagesinvitroorofmiceinvivo(Arenas-Gamboa
SciP(Fumeauxetal.,2014).PrematureaccumulationofSciPin etal.,2011;Caswelletal.,2013;Mirabellaetal.,2013).MucR(also
G2-phasehasadverseeffectsonthecellcycleduetoapremature knownasRos)regulatesvirulencegenesintheplantpathogen
shutdownofCtrA-activatedpromoterswhicharenormallyonly A.tumefaciens(Cooley,D’SouzaandKado1991;CooleyandKado
silencedwhenSciPaccumulatesinG1-phase(Goraetal.,2010; 1991)andisknowntopromotesymbiosisinRhizobiumetli(Bit-
Tanetal.,2010).TheG1-specificaccumulationofSciPseemsto tinger et al., 1997). Therefore, MucR seems to be required for
Panisetal. 129
thetranscriptionalfine-tuningofcellcyclefunctions,explain- activevariantofE.coliRelAsynthaseinC.crescentusresultsin
ingwhyitisdispensableforviability.However,MucRbindsthe stabilizationofCtrAandreducedsynthesisofDnaAintheab-
ctrApromoterregionandregulatesctrAexpressioninC.crescen- senceofstarvation(GonzalezandCollier2014).Notably,basal
tus(Fumeauxetal.,2014),indicatingaremarkabledegreeofin- (p)ppGpplevelsinC.crescentusmaintainctrAtranscriptionand
terconnectivitywithinthismoduleaspartofasystemicallyop- increasednaAtranscriptioninresponsetofattyacidstarvation
eratingregulatorycircuit(Fig.3).Addingtothiscomplexity,the (Stottetal.,submitted),incontrasttotheinhibitionofdnaAtran-
cross-regulationbythesemodulecomponentspromotesthecor- scriptionby(p)ppGppinE.coli(ChiaramelloandZyskind1990;
rectconsecutiveaccumulationoftheseregulatorsatthecorrect ZyskindandSmith1992).
timeinthecellcycleorservesaspartof(auto-)regulatorymech- Theroleofthe(p)ppGppalarmoneonthecellcycleisalso
anismthatmaintainsasteadystateasshowninFigs2and3(Fio- seen in other alpha-Proteobacteria. Induction of (p)ppGpp by
ravantietal.,2013;Murrayetal.,2013;Fiebigetal.,2014;Fumeaux carbonandnitrogenstarvationalsoyieldsG1-arrestedS.meliloti
etal.,2014). cells,aconditionthatwasrecentlyexploitedforsynchroniza-
tion (De Nisco et al., 2014). Moreover, the recent finding that
G1-arrested B. abortus cells are the predominant invasive cell
Gradualtransitionsfrommodules
typethatenterhostcells(Degheltetal.,2014)andthefactthat
If three sequential transcriptional modules regulate cell cycle B.melitensis,B.suisandB.abortusmutantsblockedin(p)ppGpp
transcripts,whyisagradualfluctuationintranscriptsobserved production are attenuated for virulence on mammalian cells
(Laubetal.,2000;McGrathetal.,2007;Fangetal.,2013),rather (Kimetal.,2005;Dozotetal.,2006)furthersuggestthatthemech-
than three transcript peaks arising from sequential transcrip- anismof(p)ppGpp-mediatedG1-arrestiscrucialtopromotepro-
tional pulses? While this question remains to be addressed ductiveinteractionsbetweenbacteriumandhost.
experimentally, several explanations can be envisioned. One Themechanismbywhich(p)ppGppaffectsproteinstability
possibility is that post-transcriptional delay mechanisms pro- iscurrentlyunknown,butcouldbemediatedbypolyphosphate
mote graduation of select transcripts. In fact, it has been (polyP)(Rao,Gomez-GarciaandKornberg2009).Theselongchain
proposed that G1-phase transcripts could be subject to post- polymersofinorganicphosphateareformedbysequentialad-
transcriptional regulation (Gora et al., 2010). Such global post- ditionofinorganicphosphatebypolyPkinaseasitconvertsATP
transcriptionalregulatorsactingonG1-phasetranscriptsremain toADP.InC.crescentus,(p)ppGpppromotespolyPaccumulation
apossibility. (Boutte and Crosson 2011). PolyP in turn has general chaper-
Alternatively,graduationoftranscriptscouldarisefromstag- one activity, protecting proteins from unfolding and aggrega-
geredtranscriptionalcontrolbynegativeregulatorsand/ordif- tion(Grayetal.,2014),modulatestheactivityoftheE.coliLon
ferentpromoteraffinitiescausingtranscriptpeakstomergeinto protease(Kurodaetal.,2001)andinfluencestheexpressionof
oneanotherupontheirinductionthrough(indirect)transcrip- thesigmafactorencodedbyrpoS(Shibaetal.,1997).Itisthus
tional hierarchies within or even across modules. Indeed the conceivablethat(p)ppGppinfluencestheabundanceofCtrAor
GcrA/CcrMmoduleinducestheexpressionofCtrA(Reisenauer DnaAindirectlythroughpolyP.
andShapiro2002;Holtzendorffetal.,2004;Fioravantietal.,2013;
Murrayetal.,2013),whichthenactivatestheexpressionofCcrM
Re-wiringtheCtrAmoduleintoquorumsensingcontrol
duringG2.CtrAalsoactsintheensuingmodulewithMucRto
restricttheexpressionofSciPandotherproteinstoG1-phase Members of the Rhodobacterales also tune CtrA abundance to
fortheconsequentdown-regulationofG2-phasepromotersby a cell-cell signaling pathway known as quorum sensing (QS)
SciP(Goraetal.,2010;Tanetal.,2010;Fumeauxetal.,2014). (Fuqua,ParsekandGreenberg2001).InQS,adiffusiblechemical
signalreleasedatbyeachbacteriumataconstantrateservesas
proxyofthenumberofcellswithinapopulation.Aseachcell
Impactof(p)ppGpponcellcycletranscription
producestheQSsignal,acriticalthresholdinthelevelofthe
It has recently emerged that cell cycle transcription is not signalisattainedwithapre-determinedconcentrationofcells.
onlyregulatedsystemically,butisalsoresponsivetosystemic QSisexecutedbyareceptorthatbindsthesignalandactivates
cues.Forexample,Crossonandcolleaguesfoundthat(p)ppGpp asignaltransductioncascadeinducingtranscriptionalchanges.
(guanosine 3(cid:4),5(cid:4)-bispyrophosphate) (Boutte and Crosson 2013), InDinoroseobactershibae,asymbioticbacteriumassociatedwith
analarmonethatisinducedunderduringcarbonandnitrogen marinealgae,ctrAexpressionisalsostronglydown-regulatedin
exhaustion, can influence the cell cycle when cells are grow- aQSmutantstrain(Patzeltetal.,2013)andacomparablemutant
inginrichmedium,i.e.underconditionswherecarbonandni- ofRuegeriasp.KLH11thatdoesnotexpressctrAisnon-motile
trogenshouldnotbelimiting(LesleyandShapiro2008;Boutte (Zanetal.,2013).ThismotilitydefectiscorrectedwhenCtrAis
andCrosson2011;Boutte,HenryandCrosson2012;Brimacombe expressedfromaconstitutivepromoterintheQSmutant,indi-
et al., 2013; Zan et al., 2013; Gonzalez and Collier 2014; Wang catingthatQSregulatesmotilitythroughCtrA(Zanetal.,2013).
etal.,2014).Atthemacroscopiclevel,(p)ppGppdelaysprogres- It is unclear how CtrA controls motility in Ruegeria sp. and in
sion from G1→S-phase in exponentially growing C. crescentus R.capsulatus,asnoCtrATTAA-N7-TTAAconsensusmotifswere
(Lesley and Shapiro 2008; Boutte and Crosson 2011; Gonzalez discernibleupstreamofmotilitygenes,suggestingthatmotil-
andCollier2014).Basallevelsofthealarmonearealsosynthe- itycontrolisindirect.As(1)aSciPorthologisencodedinthe
sizedbySpoTwhenC.crescentuscellsarestarvedforfattyacids Rhodobacteralesgenomes(Fig.1B),(2)SciPexpressionisactivated
(Stottetal.,submitted),while(p)ppGpplevelsarestronglyaug- byCtrAinC.crescentusand(3)thepredictedSciPconsensusmotif
mented in response to carbon starvation (Lesley and Shapiro overlapswithaCtrAhalfsiteinmotilitypromoters,wepredict
2008;BoutteandCrosson2011). thatSciPisaregulatorofmotilityinthesegeneraasinC.cres-
Atthemolecularlevel,(p)ppGpphasbeenreportedtoinflu- centus(Fumeauxetal.,2014).Thesituationseemstobeslightly
ence the steady-state levels of DnaA and CtrA during carbon different for D. shibae where inactivation of ctrA also leads to
starvation(LesleyandShapiro2008;BoutteandCrosson2011). down-regulation of flagellar gene expression, many of which
Artificialinductionof(p)ppGppsynthesiswithaconstitutively harboraconsensusCtrAboxesintheputativepromoters(Wang