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Fagan, R.P. orcid.org/0000-0002-8704-4828 and Fairweather, N.F. (2014) Biogenesis and functions of bacterial S-layers. Nature Reviews Microbiology, 12. 3. pp. 211-222. ISSN 1740-1526

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FaganandFairweatherpage 1

Publishedversionavailable:

http://www.nature.com/nrmicro/journal/v12/n3/full/nrmicro3213.html

BiogenesisandfunctionsofbacterialS-layers

RobertP.FaganaandNeilF.Fairweatherb

aDepartmentofMolecularBiologyandBiotechnology,UniversityofSheffield

bDepartmentofLifeSciences,MRCCentreforMolecularBacteriologyand

Infection,ImperialCollegeLondon

Correspondence:

ProfNeilFairweather,FlowersBuilding,SouthKensingtonCampus,Imperial

CollegeLondon,LondonSW72AZ

Tel:+442075945247

[email protected]


ABSTRACT

TheoutersurfaceofmanyArchaeaandBacteriaiscoatedwithaproteinaceous

surfacelayer(S-layer),whichisformedbytheself-assemblyofmonomeric

proteinsintoaregularlyspaced,two-dimensionalarray.Bacteriapossess

dedicatedpathwaysforthesecretionandanchoringoftheS-layertothecellwall

andsomeGram-positivespecieshavelargeS-layer-associatedgenefamilies.S-

layershaveimportantrolesingrowthandsurvivalandtheirmanyfunctions

includemaintenanceofcellintegrity,enzymedisplayand,inpathogensand

commensals,interactionwiththehostanditsimmunesystem.Herewereview

ourcurrentknowledgeofS-layerandS-layerassociatedproteinsincludingtheir

structures,mechanismsofsecretionandanchoringandtheirdiversefunctions.


S-layersarefoundonbothGram-positiveandGram-negativebacteriaandare

highlyprevalentintheArchaea1-3.Theyaredefinedasatwo-dimensional

crystallinearraythatcoatstheentirecellandtheyarethoughttoprovide

importantfunctionalproperties.S-layersconsistofoneormore(glyco)proteins,

termedS-layerproteins(SLPs),thatundergoself-assemblytoformaregularly

spacedarrayonthesurfaceofthecell.Assomeofthemostabundantproteinsin

thecell2,theirbiogenesismustconsumeconsiderablemetabolicresources,

reflectingtheirimportancetotheorganism.S-layerswerefirstrecognizedinthe

1950sandstudiedinnumerousspeciesduringthefollowingdecades,which

revealedconsiderabledetailoftheirstructuresusingtechniquessuchasfreeze-

etchelectronmicroscopy(seeBOX1).Suchstudies1,2,4showedstructurally

diverseS-layers,withoblique(p1,p2),square(p4)orhexagonal(p6)lattice

symmetries.SomeGram-positivespeciesharbourproteinfamiliescontaining

SLPsandSLP-relatedproteinsthatshareacommoncellwallanchoring

mechanism.Howeveritisnotyetclearifallfamilymemberscontribute

productivelytoS-layerself-assembly.Theseproteins,forexampletheBacillus

anthracisS-layerassociatedproteins(BSLs)andtheClostridiumdifficilecellwall

proteins(CWPs)(seebelow)areincludedinthisReviewasmanyare

functionallyrelevant.

ThelackofS-layersinthemodelorganismsEscherichiacoliandBacillussubtilis

hinderedtheirmolecularanalysisduringthe“molecularmicrobiology”eraofthe

1980s.Recentadvancesingenomicsandstructuralbiologytogetherwiththe

developmentofnewmolecularcloningtoolsformanyspecieshasfacilitated

structuralandfunctionalstudiesofSLPs.ComprehensivereviewsonS-layers


werewrittenoveradecadeago1,2andothershaveemphasizedtheexploitation

ofSLPsinnanotechnology1,2,56.AnexcellentreviewoftheArchaealcellenvelope

waspublishedrecently3whichincludedthepropertiesofS-layersinthisdomain

oflife.Here,wereviewthebiologyofbacterialS-layerproteinsandhighlight

recentdiscoveriesthathaveshapedourunderstandingoftheseimportant

proteins.Forconvenience,Table1outlinesalltheSLPsdescribedinthisreview.

DiversityofS-layergenesandproteins

S-layersareusuallycomposedofasingleprotein,andtheirstructuralgenescan

belinkedtogenesencodingmodificationorsecretorypathways.Despitethe

apparentlyconservedfunctionofprovidingatwo-dimensionalarray

surroundingthecell,geneticandfunctionalstudiesrevealawidediversityin

bothsequencesandrolesforS-layerproteins.

GeneticVariation

SeveralbacterialspeciesshowgeneticvariationinSLPexpression,perhapsthe

bestexampleisCampylobacterfetusinwhichS-layervariationisverywell

characterized7.InserotypeAstrainsofC.fetus,thegenomecontainsuptoeight

sapAhomologsandonepromoterelementwithina~65kbsapisland.Usually

onlyonesaphomologisexpressedinculture,althoughbacterialsub-populations

canbeidentifiedthatexpressadditionalsapproteins.Saphomologsare

expressedfromasinglesappromoter8andexhibitextensivesequence

homology.Broad,highfrequencychromosomalrearrangementsinvolvingDNA

inversionandrecombinationleadtophenotypicswitchingandexpressionof


alternatesaphomologsonthecellsurface,resultinginanantigenicallydistinct

S-layer8-10.

TheClostridiumdifficileS-layeriscomposedoftwoproteins,thehigh-molecular

weight(HMW)SLPandthelow-molecularweight(LMW)SLP,derivedby

proteolyticcleavageoftheprecursorSlpA.TheLMWSLP,whichlikelyfacesthe

environment,exhibitsconsiderablesequencevariabilitybetweenstrains11,12.

Thisvariationaffectsrecognitionbyantibodies,whichpresumablyreflects

pressurefromthehostimmuneresponse.ThegeneticbasisofS-layerdiversity

inC.difficilewasanalyzedusinghigh-throughputgenomesequencingofapanel

ofclinicallydiversestrains,whichrevealedthepresenceofa~10kbcassette

encodingslpA,secA2andtwoCWPs(seebelow)13.12divergentcassetteswere

identifiedamongthestrainsandtheauthorsproposedthatrecombinational

switchingoccursinC.difficilepopulationstogenerateantigenicdiversity.

Interestingly,oneofthecassettesissubstantiallylargerthantheothers(24kbvs

10kb)andcontains19extragenesencodingaputativeglycosylationisland(see

below).AnotherC.difficileS-layerprotein,CwpV,undergoesphase-variable

expression14mediatedbyDNAinversionofanelementsituatedbetweenthe

promoterandthestructuralgene15.

S-layergenefamilies

SomeFirmicutescontainmultipleS-layergenehomologsthatexhibitvarying

degreesofsequenceidentity,whichsuggeststhatgeneduplicationhasledtoa

familyofgeneswithfunctionaldiversity.


ThebeststudiedexamplesofSLPandassociatedproteingenefamiliesarefound

inB.anthracisandC.difficile.Basedonthepresenceofthreetandemsurface

layerhomology(SLH)motifs(seebelow)withinpredictedsurfaceproteins,24

putativeBSLswereidentifiedinB.anthracisSterne16,includingthetwomajorS-

layerproteins,SapandEA1(FIG.1).TheB.anthracisSLPsareincorporatedinto

theS-layeratdifferentstagesofgrowth;theSapS-layerisproducedduringthe

exponentialgrowthphaseandisreplacedinthestationaryphasebytheEA1S-

layer17.ThreeBSLsareencodedbyplasmids:twobypOX1andonebypOX216.

IneachBSLthethreetandemlyarrangedSLHmotifsarelocatedeitherattheN-

orC-terminusoftheprotein.Insomecasestheproteinsareconsiderablylarger

thanthe~220residuesrequiredfortheSLHdomains,theSap1andEA1

proteins,forexample,areover800residuesinlength.Insomecasesfunctional

effectordomainscanbeidentifiedintheselargerproteins,includingdomains

encodingleucinerichrepeats(LRRs),βlactamaseandseveralinvolvedin

peptidoglycansynthesisandhydrolysis.B.cereus,acloserelativeofB.anthracis,

lacksthebslG,bslKandamiAgenesofB.anthracisyetharborsthreeuniquebsl

genes,bslV,bslW,andbslX,notfoundinB.anthracis18.Itshouldbeemphasized

thattheseBSLshavenotbeenshowntoformtwo-dimensionalarrays;this

propertyisseenonlyinSapandEA1inB.anthracis.

AremarkablysimilarsituationisfoundinC.difficile.AnumberofClostridiause

theCellWallBinding2(CWB2)motifinananalogousfashiontotheSLHmotifs

toanchortheS-layertotheunderlyingcellwall(seebelow).InC.difficilewefind

29CWPseachwiththreetandemCWB2domains,includingthemajorSLP,

SlpA19.ComparisonoftheeffectordomainsassociatedwiththeB.anthracisBSL


proteinsandtheC.difficileCWPsrevealmanysimilaritiesincluding

peptidoglycanhydrolases,putativeadhesinsandLRRproteins(FIG.1).Families

ofCWB2-containingsurfaceproteins,somewitheffectordomains,arealsofound

inC.botulinumandC.tetani20,21.

ThelargenumberofSLPparalogsintheClostridiaandBacillithatcarryan

effectordomain,manyofwhicharepredictedtobeexposedtotheenvironment,

inspirestheideathattheS-layerfunctionsasascaffoldtodisplayproteinsor

glycoproteinstotheexternalenvironment.Inthisway,theS-layercanimparta

varietyoffunctionsonitshost(seebelow),dependingonthepropertiesofthe

proteinorglycoproteindisplayed.

FromproteinstofunctionalS-layers

SLPsare transported to thecell surface,where theyassemble into theordered

structuresoftheS-layers.Inaddition,tobuildafullyfunctionalS-layer,SLPsare

anchoredtothecellwallandinsomeorganismshighlyglycosylated.

SecretionofS-layerproteins

Translocationofproteinsacross the cell envelope is anessentialprocess inall

bacteria.SecretionofS-layerproteinspresentsaparticularproblemforbacteria

owing to the large quantity of protein required to form a contiguous para-

crystallinearray;forexample,weestimatethattheC.difficileS-layercontainsup

to500,000subunitsrequiring thesecretionofapproximately400subunitsper

second per cell during exponential growth. Several distinct mechanisms have


evolved to cope with this high protein flux but now, as S-layer secretion is

studiedinseveralbacterialspecies,anumberoftrendsareemerging(FIG.2).

In many Gram-negative species, including Caulobacter crescentus, Serratia

marcescens,andC. fetus, S-layer secretion relies on a specific type I secretion

system22-24 comprising an inner membrane ABC transporter and an outer

membranepore(FIG.2b).InC.fetusafour-geneoperon,adjacenttothephase-

variableS-layercassette,encodesthreeproteins,SapDEF,withhomologytotype

Isecretionsystems,andanadditionaluniqueprotein,SapC.Mutagenesisofthis

operon blocks S-layer secretion and the four gene operon is sufficient for the

secretionoftheS-layerproteinSapAinaheterologoushost24.

Inthefishpathogens,AeromonashydrophilaandAeromonassalmonicida,S-layer

secretionappearstobedependentonspecifictype II secretion systems (FIG.

2b).TheSLPsofbothspeciespossessanamino-terminalsecretionsignalandin

eachspeciesadditionalsecretionproteinswithhomologytocomponentsofthe

prototypic pullulanase secretion system, a type II secretion system found in

Klebsiella, have been identified:Aeromonas hydrophilaSpsD is homologous to

PulD andAeromonas salmonicidaApsE is homologous to PulE. Mutagenesis of

either spsD or apsE results in periplasmic accumulation of the SLPs25,26. A.

salmonicidaApsEisencodedwithinacompletetypeIIsecretionclusteradjacent

totheS-layergene,vapA.Theothergenesinthisclusterhavenotbeenstudiedin

depthbutitseemslikelythattheencodedtypeIIsecretionsystemisresponsible

forthesecretionofVapA.


S-layer secretion has been studied in detail in two Gram-positive bacteria, B.

anthracis and C. difficile (FIG. 2a). In both cases the secretion of the S-layer

precursorisdependentontheaccessorySecsecretionsystem27,28.Theaccessory

SecsecretionsystemwasfirstidentifiedinMycobacteriumtuberculosis29andhas

since been characterized in a small number of Gram-positive species30.

Organismspossessing an accessory Sec systemhave two copies of theATPase

SecA (SecA1 and SecA2); some bacteria, such as B. anthracis, also have an

accessory SecY (SecY2). The accessory ATPase, SecA2, is responsible for the

secretionofasmallsubsetofproteins.InB.anthracisefficientsecretionofboth

major S-layer proteins, EA1 and Sap, requires SecA2 and the S-layer assembly

protein, SlaP27. In C. difficile the accessory Sec system is responsible for the

secretion of the S-layer precursor SlpA and the major phase-variable cell wall

proteinCwpV28.

Interestingly, there is a striking degree of genetic linkage between the genes

encodingS-layerproteinsandtheirdedicatedsecretionsystemsinmanyofthe

organisms described above, includingC.difficile,B.anthracis,A. salmonicida,C.

fetusandC.crescentus.Thereisalsoevidenceforhorizontaltransferofthegene

cassettecontainingslpAandsecA2betweendifferentlineagesofC.difficile13.This

emphasizes the importance of the S-layer and its secretion in the life cycle of

these organisms. As molecular characterization of S-layers extends to new

species it will be fascinating to see if dedicated secretion systems and tight

geneticlinkagearecommonfeaturesofS-layerbiogenesis.

AnchoringofS-layerproteinstothecellsurface


TheS-layerisanchoredtothecellsurfacevianon-covalentinteractionswithcell

surfacestructures,mostcommonlywithLPSinGram-negativeandwithcellwall

polysaccharidesinGram-positivebacteria.Ingeneral,S-layeranchoringinGram-

negative bacteria is less well characterized than in Gram-positive bacteria.

However, the S-layers of C. crescentus and C. fetus have been studied in some

detail.InC.crescentustheN-terminal~225aminoacidsofthe98kDaRsaASLP

isrequiredforbindingtoLPSonthecellsurface31,32.Theexactmechanismofthis

interactionhasyettobecharacterized,partlybecausetheexactstructureofthe

C. crescentus LPS is unknown, but the interaction does require an intact O-

antigen31. Thehighly variableC. fetus S-layer alsobindsnon-covalently toLPS.

However C. fetus strains possess one of two distinct LPS serotypes and,

consequently,twodistinctS-layeranchoringmodules:serotypeAisexclusively

associated with a sapA-type S-layer and serotype B with a sapB-type S-layer.

EachC. fetus genome containsmultiple copies of either sapA or sapB, allowing

high-frequencyantigenicvariation(seeabove).AllSapA-typehomologueshavea

highly conserved N-terminal domain which is responsible for anchoring to

serotype A LPS. SapB-type SLPs are similar to SapA in general but have an

entirelyunrelatedN-terminaldomainwhichanchorstheproteinstoserotypeB

LPS10,33,34.

The anchoring of S-layers has been studied inmanyGram-positive species. To

date, two conserved Gram-positive S-layer anchoring modules have been

identified,utilizingeither theSLHdomainor theCWB2domain.Bothmodules

employthreedomainswhicharelocatedeitherattheN-orC-terminalregionof

theprotein.TheSLHdomain is themostwidelydistributed,beingfoundinthe


SLPs ofmanyBacillus species,Thermusthermophillus,Deinococcusradiodurans

and at least oneClostridia species,C. thermocellum35,36-40. The twomajor SLPs

producedbyB.anthracis,SapandEA1,eachhavethreetandemcopiesoftheSLH

motif 41,42. These motifs fold as a pseudo-trimer (see BOX 1)43 and act

cooperatively to bind a pyruvylated secondary cell wall polymer (SCWP).

PyruvylationoftheSCWPreliesonanenzyme,CsaB,whichisencodedadjacent

to Sap and EA1 in the B. anthracis genome40. The thermophilic bacterium

Geobacillusstearothermophiluspossessesoneofthemost intensivelystudiedS-

layers. G. stearothermophilus strains can produce five different S-layer types,

encodedbysbsA-DandsgsE44-47withtwodistinctanchoringmechanisms.SbsB

has three N-terminal copies of the SLH domain that anchor the protein to a

pyruvylated SCWP 48. However, no SLH domains can be identified in the

remainingfourGeobacillusSLPs.Instead,SbsChasbeenshowntointeractwitha

N-acetylmannosaminuronicacid–containingSCWP49viathefirst240residuesof

thematureSLP50(BOX1).AstheseresiduesarehighlyconservedinSbsA,SbsD

andSgsEitislikelythattheseSLPsareanchoredbythesamemechanism.

The second conserved mechanism involves the CWB2 motif, which was first

identifiedinCwlB–anautolysinthatcleavespeptidoglycaninthecellwallofB.

subtilis51,52. B. subtilis does not produce an S-layer but the CWB2 motif is

necessaryforretentionofCwlBinthecellwall.TheCWB2motifisfoundinmany

Clostridiaspecies,includingtheimportanthumanpathogensC.difficile,C.tetani

and C. botulinum 53. In C. difficile a family of 29 CWPs, including the S-layer

precursorSlpAandCwpV,allutilizetheCWB2domainforanchoringtothecell

wall19.Thecellwallligandforthisdomainiscurrentlyunknownbutislikelyto


beacellsurfacepolysaccharide,whichiseitherfreeorlinkedtopeptidoglycan.

We know little about the specificity of CWB2-polysaccharide interactions.

However, surface polysaccharides in the Firmicutes show considerable

diversity54, and it is possible that the CWB2 motif recognizes more than one

chemicalentity,orthatindifferentspeciesthemotifhasevolvedtorecognizea

specific polysaccharide. Each cell wall protein has three tandem copies of the

CWB2motif,analogoustothearrangementofSLHdomainsseeninotherS-layer

proteins.Asmorestructuralinformationbecomesavailableitwillbeinteresting

toseewhether thepseudo-trimerbindingarrangement isalsosharedbetween

SLH and CWB2 domains, which would suggest a common or convergent

evolutionaryorigin.

Formationofanorderedarrayonthecellsurface

S-layersarebydefinitiona twodimensionalarrayofa singleprotein,buthow

exactly is thearray formed? It is clear thatSLPs that formarrayshaveat least

twofunctionaldomains:ananchoringdomain,suchasthetandemSLHorCWB2

motifs,thatattachestheproteintotheunderlyingcellwallandacrystallization

domain thatmediates SLP-SLP interaction. Crystallizationdomains,whichmay

containseveralstructuraldomains,havebeenidentifiedinG.stearothermophilus

SbsB55andSbsC56andarepresentinSLPsofotherspeciesincludingthoseofB.

anthracis57.Inalandmarkpublication58,thethree-dimensionalcrystalstructure

of SbsB was described, showing the atomic contacts between adjacent 718

residue SLP crystallization domains and how individual structural domains

withinmoleculesareco-ordinatedbyCa2+,ananionknowntobeessentialforS-

layer formation inG.stearothermophilus (58 andseeBOX1).Thestructurealso


shows pores of approximately 30Å diameter formed at the interface between

threeadjacentsubunits,consistentwitharoleinpermeability(seebelow).

ItshouldbenotedthatnotallSLPshavebeenshownto formtwo-dimensional

arraysandlittleisknownaboutthepotentialforself-assemblyoftheassociated

proteinssuchastheB.anthracisBSLandC.difficileCWPproteins.Theseproteins

howeverarefoundwithintheS-layer,andalthoughtheyarelikelyheldinplace

byinteractionwithcellwallligands,wecannotruleoutlateralinteractionswith

therestoftheS-layer.

GlycosylationofbacterialS-layers

ThefirstdescriptionofbacterialproteinglycosylationwasintheS-layerof

Halobacteriumsalinarium59,andsincethenalargenumberofglycosylatedS-

layerproteinshavebeenidentifiedinnumerousspeciesofBacteriaandArchaea.

S-layerglycosylationhasbeenreviewedinexcellentdetailelsewhere60-62and

onlythesalientpointswillbediscussedhere.S-layerglycanmodifications

involvesugarscommonlyfoundinglycosylatedeukaryoticproteins,together

withsomeunusualsugars(60andseebelow).WhileN-andO-linkageshave

beendescribedinArchaealSLPs3,todateonlyO-linkageshavebeenfoundin

BacterialSLPs,despiteotherBacterialsurfaceproteinsexhibitingN-linked

glycans63.O-linkageinSLPsoftheBacillaceaecaninvolveserine,threonineor

tyrosine.TheoverallstructureandarchitectureoftheS-layerglycanresembles

thatofGram-negativeLPS,containingalinkageunitandupto50repeatingunits,

eachconsistingof2-6sugars.Thisresemblancesuggestsacommonevolutionary

originofLPSbiosynthesisandS-layerglycosylation64,anideafurther


strengthenedbyrecentdescriptionsoftheS-layerglycan(slg)geneclustersinG.

stearothermophilus,Paenibacillusalvei,Geobacillustepidamans,Aneurinibacillus

thermoaerophilusandTannerellaforsythiaencodingtheglycosyltransferases,

glycanprocessingenzymesandmembranetransportmachinerysufficientfor

glycanbiosyntheticpathways(forareview,see60).S-layerglycosylation

pathwayshavebeendescribedinseveralspecies,includingG.

stearothermophilus65,P.alvei66andT.forsythia67,leadingtotheproposalofa

biosyntheticrouteinvolvingtransferofgalactosefromthenucleotide-activated

sugarUDP-α-D-Galtoalipidcarrier,formationofthelinkageunitbyadditionof

glycansandassemblyofthegrowingrepetitiveglycanchainontothelinkage

unit.Thesereactionsoccurinthecytoplasmpriortotransportofthecompleted

glycanchainviaanABC-transporter(inthecaseofG.stearothermophilus)tothe

distalsideofthemembranewheretheglycanisligatedtotheS-layerprotein

substrate60.TheglycanchainsdecoratingS-layerproteinsarefairlydiverse:for

example,inG.stearothermophilustheglycanchainisasimplepolymerofL-

rhamnose,inP.alveiL-rhamnose,N-acetylmannosamine,D-glucoseandD-

galactosearefoundandinT.forsythiatheunusualsugarsN-

acetylmannosaminuronicacid,5-acetimidol-7-N-glycolylpseudaminicacidand

digtoxosearepresent67.Whethertheglycanchainisco-transportedwiththeS-

layerproteinsubstrateremainstobedetermined,butproteintransport

(secretion)isnotdependentonglycosylation.

Recently,strainsofC.difficileweredescribedthatcontainaputativeslglocus

adjacenttoslpA13.ThisbacteriumdoesnotnormallyelaborateaglycosylatedS-

layer68butthesevariantstrainscontainadistinctS-layercassette(seeabove)


andproduceS-layerproteinsofreducedpolypeptidelength12,13.Whetherthese

strainsdoindeedhaveaglycosylatedS-layerandwhat,ifany,phenotypethat

mightconferiscurrentlyunknown.

FunctionalheterogeneityofS-layers

Itisperhapsnotsurprisingthat,asthemajorproteinaceoussurfacecomponent

ofthecell,avarietyoffunctionshavebeendescribedandproposedfortheS-

layer1,69(FIG.3).However,afterdecadesofresearch,nosinglefunctioncanbe

ascribedtotheS-layerandinmanyspeciestheS-layerhasnoknownfunction.

Theabilitytoformatwo-dimensionalarrayappearstobetheresultof

convergentevolutionandisseeninproteinsofquitedistinctsequence.SLPsand

associatedproteinshavefurtherevolvedtoadoptamultitudeofactivities,some

essentialtothephysiologyofthecellandothersfacilitatingsurvivalinspecific

niches.AlthoughwedonotdefinitivelyknowthefunctionofmanyS-layers,itis

clearfromtheirwideoccurrenceintheBacterialkingdom,apparentconvergent

evolutionandtheenormousmetabolicloadrequiredtoproduceandmaintain

thesestructuresthattheyfulfilsomeimportantroleforthebacteriathat

producethem.

RolesoftheS-layerinpathogenesisandimmunity

InLactobacilluscrispatus,anindigenousmemberofthehumanandchickengut

microflora,theS-layerCsbA(SlpB)hasanN-terminaldomainthatbindstypesI

andIVcollagenandaC–terminaldomainthatinteractswiththebacterialcell

wall70.Thiscollagenbindingactivityisthoughttomediatebacterialcolonization


ofthegut(FIG3a).Interestingly,twootherputativeS-layergenesarepresentin

thisstrain:slpC,locateddownstreamofcsbA,istranscriptionallyactiveandslpA

issilent70-72.

AsthemajorsurfaceantigenofC.difficile,theS-layerhasbeeninvestigatedfor

itsabilitytoberecognizedbyandactivatetheimmunesystem.TheSlpAproteins

(HMWandLMWSLPs)inducereleaseofpro-inflammatorycytokinesfrom

humanmonocytesandinducematurationofhumanmonocyte-deriveddendritic

cells(MDDCs)73.FurtherworkadvancedthesefindingsbyshowingthattheSLPs

inducedmaturationof mouse bone marrow derived dendritic cells and the

production of the cytokines IL-12, TNFα and IL-10, but not of IL-1β. Importantly,

activation of dendritic cells was dependentonToll-likereceptor4(TLR4)and

subsequentlyinducedThresponsesknowntobeinvolvedinclearanceof

bacterialpathogens74.InfectionofTLR4knockoutmiceresultedinincreased

severityofsymptomscomparedtowild-typemice,suggestinganimportantrole

ofTLR4inbacterialclearance74.BoththeHMWandLMWSLPsofC.difficilewere

requiredfordendriticcellactivation,suggestingeithertheentirecomplexis

recognizedbyTLR4,orthatoneoftheSLPsistheligand,butrequirestobeseen

inthecontextoftheHMW-LMWcomplex(FIG.3a).SlpAhasalsobeenshownto

bindtofixedgutenterocytes75,whichcouldberelatedtoanimmunestimulatory

roleandinfersthattheS-layermightfunctionasanadhesininvivo(FIG3a).To

date,theroleofSlpAasanadhesinhasnotbeeninvestigatedthrough

mutagenesisasslpAisanessentialgene,butrecentadvancesingenetics76-78

shouldallowcreationofconditionalmutantstrainsthatcouldbeusedin

infectionexperimentstotestthishypothesis.Finallythephase-variableCwpV


proteinhasbacterialauto-aggregationactivitythatmighthavearoleduring

infection15.

InB.anthracis,thepreciseroleofSapandEA1areunknown,butactivitiesof

someBSLshavebeenelucidated.TheBslAproteinhasbeenshowntomediate

adhesionofencapsulatedB.anthracistoHeLacells16.bslAmutantsdisplay

limiteddisseminationtotissuesanddecreasedvirulenceinaguineapigmodelof

infection,suggestingBslAisafunctionaladhesinrequiredforfullvirulenceofB.

anthracis79.AnotherS-layerprotein,BslK,mediateshemeuptakebyutilizinga

nearirontransporter(NEAT)domain80(FIG3c).InB.cereus,whichhashighly

similarBSLstoB.anthracisbutaverydifferentcapsulecomposition,acsaB

mutantwasfoundtobedefectiveinretainingSap,EA1andBslOonthecell

wall18.ThecsaBmutantexhibitedreducedvirulenceinamousemodelof

infection,implicatingoneormoreBSLsinthepathogenesisofanthrax.

TheGram-negativepathogenC.fetusisaleadingcauseofabortionsinsheepand

cattleandcancausepersistentsystemicinfectionsinhumans.TheS-layer,

composedoftheSapprotein,isessentialforhostcolonization81.Sapis

antigenicallyvariable(seeabove)whichcontributestotheevasionofthehost

immuneresponse82,83.TheS-layeriscrucialforpathogenesisasmutantstrains

donotcausediseaseandaresensitivetophagocytosisandkillinginserum

mediatedbycomplementC384-86(FIG3a).Antigenicvariationoccursduring

infectioninhumans,asshownbystrainsisolatedatearlyandlatestagesof

infectionfromfourindividualswithrelapsingC.fetusinfections87.Inthree

patients,thestrainhadundergoneaswitchinthepredominantS-layer


expressed.

T.forsythia(previouslyknownasBacteroidesforsythus)isaGram-negative

anaerobethatisassociatedwithsevereformsofperiodontaldisease88,89.Strains

ofT.forsythiaproducetwohighmolecularweightproteins,eachover200kDa,

thatareconcomitantlyexpressedandconstitutetheS-layer90-92.Thestructural

genestfsAandtfsBencodeproteinsof120and140kDa93,suggestingpost-

translationalmodification.Thiswasexploredindetailinastudy67thatshoweda

complexpatternofglycosylationonbothproteinsincludingamodified

pseudaminicacidPse5Am7Gcnotpreviouslyfoundonbacterialproteins.This

sugar,asialicacidderivative,wassuggestedtoparticipateinbacterium-host

interactions67,basedontheprevalenceofsialicacid-likesugarsinGram-

negativestructuresinvolvedinpathogenesissuchasLPS,capsules,piliand

flagella.InT.forsythiatransposonmutantsthatexhibitedalteredbiofilm

formation(FIG3c),anoperoninvolvedinexo-polysaccharidebiosynthesiswas

identified.Mutationofonegene,weeC,whichencodesaputativeUDP-N-acetyl-

D-mannosaminuronicaciddehydrogenase,increasedbiofilmformationand

alteredthemobilityofthetwoT.forsythiaS-layerproteinsonSDS-PAGE,an

effectconsistentwithglycosylation94.Aproteomicsstudyalsofoundincreased

quantitiesofS-layerproteinsinTannerellabiofilmscomparedtoplanktonic

cells95.TheT.forsythiaS-layerappearsessentialforvirulenceofthispathogenas

adhesiontoandinvasionofhumangingivalepithelialcells(Ca9-22cells)and

mouthepidermalcarcinomacells(KBcells)weredecreasedorabolishedina

mutantdefectiveintfsAandtfsB92.

SLPshavealsobeendemonstratedtohavearoleinprotectionagainstpredation.


TheparasiticbacteriumBdellovibriobacterovirushasawidehostrangeand

infectsandreplicatesintheperiplasmofsusceptibleGram-negativespecies96.

StrainsofAquaspirillumserpensandCaulobactercresentuspossessanS-layer

andarenotnormallyparasitizedbyBdellovibrio,butS-layernegativevariantsof

bothspecieswereshowntobesensitivetopredation,indicatingthattheS-layer

canprovideaprotectivecoatagainstinfectionwithBdellovibrio97(FIG3a).In

additiontothis,onemightspeculatethattheS-layerwouldactasareceptorfor

bacteriophageandbacteriocins,buttoourknowledgethereisnoevidencefor

suchactivities.

Permeabilityandbiogenesisofthecellenvelope

S-layersareoftenproposedtofunctionapermeabilitybarrierandadirectrole

forSLPsasbarriershasbeeninvestigatedinB.coagulansandotherspecies2,98.

Experimentallydeterminedexclusionlimitsofisolatedsacculiandassociated

proteinsof15to34kDaareconsistentwithporediametersof20–60Åwithin

theS-layerlattice2,98andthecrystalstructureofSbsBrevealsporesof

approximately30Å58.ThusarolefortheS-layerasapermeabilitybarrier(FIG

3b)iscertainlyconceivable,butourknowledgeofthisactivitywouldbe

strengthenedbygeneticanalysisandatomic-resolutionimagingofalterationsin

poresizeinvivo.

InDeinococcusradiodurans,theS-layer,(knownashexagonallypacked

intermediate(HPI)layer)iscomposedoftwoS-layerproteins,SlpAandHpi,in

additiontolipidsandcarbohydrates.DeletionofslpAresultsinmajorstructural

alterationssuchaslossoftheHpiproteinandsurfaceglycans,whichcanbe


visualizedbyelectronmicroscopyaslayersofmaterialpeelingoffthecellwall99.

ThissuggeststhatSlpAisinvolvedintheattachmentofHpiandother

componentstotheunderlyingcellwall,whichisconsistentwiththepresenceof

SLHdomainswithinSlpA.OtherexamplesofarolefortheS-layerincell

envelopebiogenesisincludeC.difficile,inwhichinhibitionofcleavageoftheS-

layerprecursorSlpAthroughinactivationoftheproteaseCwp84leadsto

incorrectassemblyoftheS-layerandsheddingoffull-lengthSlpAfromthecell

wall100,101(FIG3b).Interestingly,virulenceofthiscwp84mutantisnot

diminishedinthehamstermodelofinfection,presumablybecausegutproteases

suchastrypsincancleaveSlpAresultinginafullyfunctionalS-layer100.

InBacillusanthracis,thephysiologicalfunctionsoftheSap,EA1andBslproteins

havebeeninvestigated.SapandEA1bothhaveapeptidoglycanhydrolase

activity102,althoughtheylackknownfunctionaldomainsthatspecifysuchan

activity.BslO,whichhasputativeN-acetylglucosaminidaseactivityandis

localizedatthecellsepta,hasaroleincatalyzingcelldivisionwithbslOmutants

exhibitingincreasedlengthsofcellchains103.(FIG3c).Elongatedchainsof

bacteriaarealsoseeninasapmutant,andthislackofSapcanbecomplemented

bytheadditionofpurifiedBslOproteintotheculturemedium,restoringcell

divisionandreducingthechainlength.Inwild-typecells,Sapwasvisualized

predominantlyonthelateralcellwall,awayfromthesepta,suggestingthatB.

anthraciscontrolsthespatialdepositionoftheSLPsinordertoensurecorrect

localizationoffunctionalentitiessuchasBslO104.IntheC.difficilefamilyof

CWPs/SLPs,severalproteinspotentiallymediatepeptidoglycansynthesisand

remodeling,includingCwp22whichhasL,D-transpeptidaseactivity105.!


OtherfunctionsoftheS-layer

AnotherfunctionassociatedwiththeS-layerincludesswimminginthemarine

bacteriumSynechococcus106(FIG3c).Thisspeciesishighlymotilebutdoesnot

possessanyobviousflagellumorotherorganellesthatmightmediateswimming,

andthemechanicalbasisforitsmotilityislargelyamystery.Inmutants

defectiveforswimmingtwosurfaceproteinsessentialforthisactivitywere

identified:SwmAandSwmB107.SwmAisa130kDaglycosylatedS-layerprotein

andSwmBalarge1.12mDaprotein.Othergenesessentialforswimmingencode

anABCtransporterandseveralglycosyltransferases108.Althoughtheseresults

indicateanotherfunctionforanS-layerandsuggestglycosylationofSwmAis

requiredformotilitytheydonot,unfortunately,leadusmuchfurtherin

elucidatingthishighlyunusualmechanismofswimming.

Perspectives

Followingtheirdiscoveryinthe1950sandafterdecadesofresearch,our

knowledgeofBacterialSLPshasincreasedconsiderablyinthelastfewyears.Itis

clearthatS-layersdonothaveonesinglefunction,ratheradiversityoffunctions

isapparentandweexpecttoseenewfunctionsrevealedasmorespeciesare

studied.InsomeArchaea,forexampleSulfolobus,theS-layerappearstobethe

solenon-lipidconstituentoftheenvelope3,suggestingstructuralintegritymight

beanancestralfunctionofSLPs.Insomebacterialspecies,suchastheClostridia,

theS-layerappearsessentialforcellviability,asunconditionaldeletionmutants


cannotbeconstructed.Inthesespecies,theSLPisthemainproteincomponent

ofthecellsurfacealthoughadiversityofothermacromoleculesisfound.

Increasinglysophisticatedandhigh-resolutiontechniquessuchasAFMand

electroncrystallographyarebeingappliedtostudyS-layermorphologyand

symmetry109.Ultimatelythesetechniqueswillbecombinedwithstructural

informationfromX-raycrystallographyorNMRtogenerateatomicresolution

modelsofthecompleteS-layer.Recently,progresshasbeenmadewithatomic

resolutionstructuresofseveralSLPs,andwelookforwardtoacomplete

descriptionofanassembledS-layerstructureincomplexwiththeligand

responsibleforanchoringtothecellwall.Onlythenwillwebeabletoaddress

thecrucialoutstandingquestionsinS-layerbiology:whatisthebiochemical

basisofparacrystallinearrayself-assembly?Whatmechanismsareemployedto

attachtheS-layertothecellsurface?Andfinally,whatisthestructuralbasisfor

theknownS-layerfunctions?

ItisclearthatSLPsandtheirassociatedproteinshaveevolvedspecialized

functions,andinsomespeciesofFirmicutesSLPsactasascaffoldtodisplay

enzymesonthecellsurface.ItislikelythatmanymoreSLPswillbeidentified

fromgenomesequencinganditwillbeachallengetoassignmeaningful

functionstothisdiversefamilyofproteinswithoutlaboratoryinvestigation.

PrioritiesforfutureresearchincludeestablishingthefunctionsofS-layers

presentinbacterialpathogens,investigatingtheirpotentialastherapeutic

targetsforantimicrobialorvaccinedevelopment,andindepthstructural

analysisoftheinteractionsbetweenS-layersandothersurfacecomponents.


Withtheavailabilityofincreasinglysophisticatedstructuralandimagingtools,

wearenowinapositiontopushforwardBacterialS-layerresearchandperhaps

determinethefullcontributionofthesefascinatingstructurestothegrowthand

survivalofBacteriawhichproducethem.


Table1|SummaryofSLPsdescribedintext

Organism SLPs Features

Campylobacterfetus SapA HighfrequencyantigenicvariationofS-layerthroughrecombinationalswitchingofsaphomologues;secretedbyaspecifictypeIsecretionsystem

SapB

Clostridiumdifficile SlpAandtheCWPfamily

SlpAessentialforcellgrowth;S-layerfunctionalisedbydecorationwithupto28additionalCWPs;secretedbytheaccessorySecsystem;mediatesinteractionswithepithelialcellsandactivatesdendriticcells

Bacillusanthracis Sap,EA1andtheBSLfamily

SapandEA1arealternateSLPs;S-layerfunctionalisedbydecorationwithBSLs;secretedbytheaccessorySecsystem;anchoredviainteractionwithpyruvylatedSCWP

Caulobactercrescentus RsaA SecretedbyaspecifictypeIsecretionsystem;anchoredviainteractionwithLPS

Aeromonassalmonicida VapA SecretedbyadedicatedtypeIIsecretionsystem

Geobacillusstearothermophilus SbsA AnchoredviainteractionwithpyruvylatedSCWP(SbsB)orN-acetylmannosaminuronicacid(SbsA,C,DandSgsE);Glycosylated

SbsB SbsC SbsD SgsETannerellaforsythia TfsA Glycosylated;S-layerincludesbothSLPs;

glycosylationrequiredforbiofilmformation;S-layeressentialforvirulence

TfsB

Lactobacilluscrispatus CsbA MediatesbindingtotypesIandIVcollagen(CsbA) SlpA

SlpCDeinococcusradiodurans SlpA S-layerincludesbothSLPs;playsarole

inmaintenanceofenvelopeintegrity HpiSynechococcus SwmA Glycosylated;requiredforswimming

motility


Figure1|ClostridiumdifficileandBacillusanthraciscellsurfaceprotein

families.DomainidentificationandorganizationamongallmembersoftheC.

difficileCWB2familyandtheB.anthracisSLHfamilywasdeterminedusingthe

Pfamproteinfamiliesdatabase120andoutlinedinSupplementaryTable1.TheN-

terminalsignalpeptide,whichisremovedupontranslocationthroughtheSec

membranechannel,isshownasablackbox,seealsoFigure2a.InC.difficilethe

secretionofatleastSlpAandCwpVisdependentontheaccessorySecsecretion

system28.InB.anthracisthetwomajorS-layerproteins,SapandEA1,also

requiretheaccessorySecsystemforsecretion104.Althoughlimiteddatais

available,itispossiblethatsecretionviatheaccessorySecsystemisacommon

featureofthesetwoproteinfamilies.


Figure2|SecretionofBacterialS-layerproteins.Todate,S-layersecretion

hasbeenstudiedinasmallnumberofspecies,inwhichanumberofdedicated

secretionsystemshavebeenidentified.a|IntheGram-positivebacteria

ClostridiumdifficileandBacillusanthracissecretionoftheS-layerprecursorsis

mediatedbytheaccessorySecsecretionsystem30.TheproteinscontainanN-

terminalsignalsequence(whitebox)whichdirectsthenascentpolypeptideto

thesecretionapparatusandiscleaveduponmembranetranslocation(indicated

withtheblacktriangle).InbothC.difficileandB.anthracis,translocation

requirestheaccessoryATPase,SecA227,28.FollowingrecognitionbySecA2,the

nascentpolypeptideistranslocatedacrossthemembranethroughapore

consistingofSecYEG(C.difficile)orSecY2EG(B.anthracis).b|SecretionoftheS-

layerproteins(SLPs)inAeromonassalmonicidaandAeromonashydrophila

requiresadedicatedTypeIIsecretionsystem25,26.TypeIIsecretionisatwo-step

process:theunfoldedprecursorisfirsttranslocatedacrossthecytoplasmic

membranebythecanonicalSecsecretionsystem,theproteinthenfoldsandis

transportedacrosstheoutermembranebyacomplexmulti-proteinsecretion

apparatuswhichiscloselyrelatedtotypeIVpili121.AcompletetypeIIsecretion

systemisencodedalongsidevapAinA.salmonicidabutonlyonecomponentof


thissystemhasbeendirectlylinkedtoSLPsecretion;A.salmonicidaApsEis

homologoustothesecretionATPase,PulE.APulDhomologue,SpsD,likely

forminganoutermembranepore,hasalsobeenidentifiedinA.hydrophila.

FurtheranalysisisrequiredtoconfirmwhetherornotSpsDandApsEarefrom

thesameconservedsecretionsystem.InCampylobacterfetustheSLPsare

secretedinasinglestepbyaTypeIsecretionsystemencodedbySapDEF24.Type

IsecretioninvolvesaninnermembraneABCtransporter,amembranefusion

proteinandanoutermembranepore.Whereknown,theanchoringdomainsof

theSLPsarehighlightedashatchedboxes,seeFigure1.


Figure3|FunctionsofS-layerproteins.

Probablerolesininfection(panela)includeadhesinactivity,foundinseveral

species:BslAofB.anthracisbindstoHeLacellsandmutantsareattenuatedin

modelsofinfection79,bindingofSLPstoentericcellshasbeenobservedinC.

difficile75andtodefinedligands(typesIandIVcollagen)inLactobacillus

crispatus70.InC.difficile,interactionofSlpAwithhostTLR4receptorsislinkedto

innateimmunity74andinC.fetus,SLPspreventbindingofcomplementfactor

C3b,protectingthebacteriumfromhostmediatedphagocytosisandserum

killing86.AroleinresistancetopredationhasbeendemonstratedinBdellovibrio

bacteriovirus97andpotentialrolesinresistancetobacteriophageand

bacteriocinsarepossiblebutremainspeculative(panela,bottom).SLPshave

rolesinmaintenanceofcellenvelopeintegrity(panelb)inDeinococcus

radioduranswhereinactivationofslpAcausessheddingofsurfacemolecules99

andinC.difficilewherelossofcwp84resultsinanabnormalS-layerand


sheddingofsurfaceproteins101.B.anthracisBslOandtheC.difficileCwp22have

peptidoglycanhydrolaseactivitythatmayremodelthepeptidoglycan.Arolefor

SLPsasapermeabilitybarrierhasbeendemonstratedinBacilluscoagulans98.A

roleincelldivisionhasbeenfoundinB.anthracis(panelc)whereinactivationof

BslO,aputativeN-acetylglucosaminidase,resultsinincreasedlengthsofchains

ofbacteria103.SLPshaverolesinaggregation(C.difficileCwpV15),biofilm

formation(T.forsythia94)andswimming(Synechococcus106).FinallyBslKfromB.

anthracismediatesironuptakebyscavengingheme,transferringittothesurface

proteinIsdC80.


Box1

SincethefirstreportedobservationofanS-layerin1953110increasingly

sophisticatedandhigh-resolutiontechniqueshavebeenappliedtothestudyof

theirmorphology,symmetryand,ultimately,atomicstructure.Someofthemost

strikingearlyvisualizationsofS-layermorphologycamefromelectron

microscopyofnegatively-stainedcellwallfragmentsandisolatedS-layersand

freeze-fracturemicroscopyofintactcells4,111.S-layerproteins(SLPs)

spontaneouslyform2-dimensionalcrystalsinvitro,whichcanbestudiedusing

electronmicroscopy(panelAshowstheregularhexagonalsurfacearrayofa

Thermoanaerobacterthermohydrosulfuricuscell112).Moredetailedstructural

informationcanbeobtainedfromtwo-dimensionalcrystalsusingelectron

crystallography(forexampleAcetogeniumkivui,transmissionelectron

microscopyimagesofnegativelystainedS-layerfragments(panelB,top),the

resultingelectrondiffractionpatterns(panelB,top,insets),3Dprojectionmap

showninpanelB(bottom)).Theprojectionmapclearlyshowsthep6hexagonal

symmetryoftheS-layerandthe6individualSLPmonomerswhichformthe

ring-likecorecomponentofthesupramolecularstructure113.Inrecentyears,

atomicforcemicroscopy(AFM)hasbeendevelopedasahighresolutionimaging


techniquetovisualizemacromoleculesandisparticularlywellsuitedtothe

studyofbacterialsurfaces114,asitallowsunprecedentedresolutiononintactS-

layersamplesundernativeaqueousconditionsand,withfunctionalisedtips,the

abilitytomapindividualproteinswithinthelayer.(PanelC,AFMimageofthe

re-assembledLysinibacillussphaericusSbpAS-layershowingcleartetragonal

symmetry115).Owingtodifficultiesingrowing3DcrystalssuitableforX-ray

crystallography,whichisinpartattributabletothepropensityofSLPstoform

two-dimensionalcrystals,thereisadearthofhighresolutionS-layerstructures.

Manygroupshavetakenadivideandconquerapproachbycrystallizingexcept

fromstructuresofrecombinantorproteolytically-derivedfragmentsofSLPs.

Thefirsttobepublishedwasa52residuecoiled-coilfragmentofthe

tetrabrachionSLPfromtheextremethermophileStaphylothermusmarinus116

followedbytwopartialstructuresofArchaealSLPsfromMethanosarcina

spp.117,118.Onecomprisesanovelseven-bladedșpropeller(theYVTNdomain),

apolycystickidneydiseasesuperfamilyfolddomainandathird,asyet

unstructured,domainwhichispredictedtoadoptaparallelright-handedș

helixfold.TheotherSLPhastwohighlyrelatedDUF1608domains,oneofwhich

hasapairoflinkedșsandwichfolds,andatransmembraneanchor118.Partial

structureshavealsobeensolvedfortwooftheGeobacillusstearothermophilus

SLPs(SbsClackingtheC-terminalcrystallizationdomain55,andSbsBlackingthe

N-terminalSLHdomains58)andC.difficileLMWSLP119.SbsCanchorstothecell

surfacevianon-covalentinteractionsbetweendomain1oftheprotein(residues

31-270)andanegativelychargedsecondarycellwallpolymer(SCWP)(seemain

text).Thecrystalstructureofthisdomainrevealedaseriesofsurfaceexposed


positivelychargedresidues,withspacingapproximatingthatofthenegatively

chargedManNAcUAgroupsontheSCWP,aswellasapotentialcarbohydrate-

bindingstackofaromaticsidechains.Furtherresearchwillhopefullydetermine

theexactcontributionoftheseresiduestobindingtheSCWP.SbsBwasthefirst

SLPtobecrystallizedinanintactform,employingnanobodiestoinhibit2D

latticeformationandallow3Dcrystallization.ThecellwallbindingSLHdomains

didnotresolveinthefinishedstructurebutthecrystalpacking,incombination

withcryo-EMandcrosslinkingstudies,allowedtheproposalofaplausiblemodel

ofthecomplete2Dparacrystallinearray(seepanelDandtext)58.The3D

structureoftheSLHdomainsfromanotherSLP,theB.anthracisSap1,hasbeen

determined,revealingapseudo-trimerwitheachSLHdomaincontributingone

componentofathreeprongedspindle,allowingmodellingofbindingoftheSLH

domainstotheSCWP43.PanelsA,BandCarereproducedwithpermissionfrom

112,113and115respectively.PanelD:thestructuralcoordinatesforSbsB32–920

(4AQ1)58weredownloadedfromthePDB(http://www.rcsb.org/pdb)andthe

imageshownwasgeneratedusingPyMOL.


SIDEBARSNOTES

Phase-variableexpression–randomvariationofgeneexpressioninabacterial

populationinwhichexpressioninindividualcellsiseitheronoroffleadingto

phenotypicheterogeneityinthepopulation.

SecondaryCellWallPolymers–carbohydratebasedpolymersotherthan

peptidoglycanandanionicpolymerspresentinthecellwall,forexamplethe

pyruvylatedB.anthracisSCWPthatanchorstheSLPsEA1andSaptothecell

wall.

N-andO-linkedglycosylation–linkageofasugartothenitrogen(N)atomof

asparagineortotheoxygen(O)atomofserine,threonineortyrosine.

TypeIsecretion-asec-independentproteinsecretionsysteminGram-negative

bacteriaconsistingofaninnermembraneABCtransporter,aperiplasmic

membranefusionproteinandanoutermembranepore.

TypeIIsecretion–asec-dependentmulti-proteinsecretionsysteminGram-

negativebacteriawhichiscloselyrelatedtotypeIIpili.

Sacculi–thesacculus(singular)isthesacofpolymerisedpeptidoglycan

surroundingthebacteria.Isolatedfromthebacterium,itretainstheshapeofthe

cell.


Acknowledgments:workintheN.F.laboratoryiscurrentlysupportedbythe

MRC(grantsG0800170andG1001721),theWellcomeTrust(grant090969Z)

andtheLeverhulmeTrust(grantRF2012-232),andintheR.F.laboratorybythe

UniversityofSheffield.

Competinginterestsstatement.Theauthorsdeclarenocompetingfinancial

interests.


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