1

‘Add,stirandreduce’:TheYersiniaeasmodelbacteriafortheevolutionof1

mammalianpathogens2

3

AlanMcNally1*,NicholasRThomson2,3,SandraReuter3,4&BrendanWWren24

5

6

1PathogenResearchGroup,NottinghamTrentUniversity,CliftonLane,7

NottinghamNG118NS8

2DepartmentofPathogenMolecularBiology,LondonSchoolofHygieneand9

TropicalMedicine,KeppelStreet,London,WC1E7HT10

3PathogenGenomics,WellcomeTrustSangerInstitute,Hinxton,CambsCB101SA11

4DepartmentofMedicine,UniversityofCambridge,Box157Addenbrooke’s12

Hospital,HillsRoad,CambridgeCB20QQ13

14KeyPoints:15

• TheevolutionofmammalianpathogenesisintheYersiniagenushas16

occurredinaparallelfashionviaabalancedmixtureofgenegainandgeneloss17

events18

• Onlybysequencingpathogenicandnonpathogenicrepresentatives19

positionedacrossanentirebacterialgenuscansuchobservationsbemade20

• Theparallelnatureoftheevolutionofpathogenesisextendsbeyond21

Yersiniaandintootherentericpathogens,notablythesalmonellae22

• Genelosseventsleadtonicherestrictionduetoareductioninmetabolic23

flexibility,oftenseeninthemoreacutelypathogenicmembersofalineage24

2

• Potentialnegativeimpactsofgenegaineventsareoffsetbythe1

transcriptionalsilencingorfinecontroloftheseacquiredelementsbyancestral2

regulonssuchasRovAandH-NS. 3

3

Abstract1

Inthestudyofmolecularmicrobiologyandbacterialgenetics,pathogenicspecies2

oftheYersiniagenushavebeenpillarsforresearchaimedatunderstandinghow3

bacteriaevolveintomammalianpathogens.Theadventoflarge-scalepopulation4

genomicstudieshashugelyacceleratedprogressinthisfield,andthepathogenic5

Yersinia species have re-emerged as model organisms to help shape our6

understanding of the evolutionary processes involved in pathogenesis. In this7

review, we highlight how microbial genomic studies of the yersiniae have8

revealeddistinctfeaturesmarkingtheevolutionarypathtowardspathogenesis,9

whicharechangingourunderstandingofpathogenevolution.Asthesefeatures10

arealsofoundinthegenomesofothermembersoftheEnterobacteriaceae,they11

provideablueprintfortheevolutionofenteropathogenicbacteria.12

13

4

Introduction1

The ever-expanding microbial genome sequence dataset, combined with an2

increasedunderstandingofthepathogenesisandecologyofbacterialpathogens,3

is illuminating our view of the dynamics that have shaped the evolution of4

bacterial pathogens. It is now evident that many bacterial pathogens infect5

humans only incidentally and often produce virulence factors that are active6

againstnon-mammalianadversariesasdiverseasinsects,protozoa,nematodes,7

predatory bacteria and bacteriophage1–3. These hosts provide a considerable8

driving force in theevolutionofbacterialpathogensenablingus to re-evaluate9

human—pathogen interactions in the light of bacterial ecology and evolution10

(theeco-evoperspective).Aconsequenceoftheeco–evoperspectiveisthatitis11

notsurprisingthatgenesencoding‘virulencefactors’arefoundinpathogensand12

non-pathogens colonizing diverse mammalian and non-mammalian hosts4,5.13

Conversely, as bacteria evolve to inhabit new hosts, or new nicheswithin the14

samehosts,usinggenomicswecanseeremnantsofgenesandgeneclustersthat15

arethoughttohaveprovidedanadaptiveadvantageinthepast,butthatarenow16

non-functionalandrepresentevolutionarybaggage.17

Thus,manybacteriaseemtobepre-armedwithvirulencedeterminantsasthey18

traverse new environments and niches, and their evolution is not solely19

dependent on mammalian/human–pathogen interactions6,7. The yersiniae20

representagenusforwhichtheeco-evoperspectiveishighlypertinentandfor21

which all 18 species in the genus have recently been fully sequenced,22

metabolically phenotyped and compared8. The genus includes both non-23

pathogenicandpathogenicspecies,andadiversityofpathogenesisphenotypes24

soitrepresentsakeymodelforunderstandingtheforcesatplayintheevolution25

5

ofpathogenicbacteria.OurcurrentunderstandingoftheYersiniaencompassesa1

long-term view of how broad host range pathogenic species such as Yersinia2

enterocolitica evolveovermillionsofyears fromanon-pathogenicancestor9. It3

also encompasses short term evolution examining how recent evolutionary4

bottlenecks have led to rapid emergence of high-pathogenic clones from their5

ancestral lineage such as the emergence of Yersinia pestis from Yersinia6

pseudotuberculosis10.7

The evolution of pathogenic Yersinia spp. shows remarkable parallels with8

membersofthegenusSalmonella,whichencompassesthebroadhostrangeself-9

limitingdiarrheagenicSalmonellaentericasubsp.entericaserovarTyphimurium10

(S. Typhimurium) as well as those that are host restricted and acutely11

pathogenic,suchasSalmonellaentericasubsp.entericaserovarTyphi(S.Typhi).12

ThishasalsomadethepathogenicYersiniaspeciesorganismsofchoiceformuch13

of the pioneeringwork performed understanding themechanisms of bacterial14

pathogenesisthroughclassicalgenetics.StudiesusingY.enterocoliticahavebeen15

at the forefront of developing our understanding of how bacteria invade host16

epithelial cells11. Studies of the pathogenic Yersinia also forged our17

understandingoftheroleofplasmidsinbacterialvirulence12,13,theidentification18

and characterization of Type III secretion systems and their role in host19

subversion14.Thisinturnledtoadvancesinourunderstandingofhowbacteria20

evadehostresponsesduringinfection15–17.21

In this Review,we examine how the eco-evo perspective, informed bywhole-22

genome sequencing and by population genomic and ecological studies, has23

challengedprevioushypothesesontheevolutionofmammalianpathogenesisin24

Yersiniaspp.Furthermore,weconsiderhowthesestudiesarerevealingcommon25

6

principles in the emergence of pathogenesis among other Enterobacteriaceae.1

Finally,weshowhowconsideringallmembersofagenusinpopulationgenomic2

studies, and not just pathogenic species, can provide greater insight and3

resolution for understanding of the processes underpinning the evolution of4

mammalianpathogenesis.5

6

Theyersiniae7

The Yersinia is a genus of Gram-negative bacteria belonging to the8

Enterobacteriaceae. The genus nomenclature is based on classical systematics9

and biochemical speciation methods, resulting in a highly diverse group of10

bacteria with 18 defined species18–26. The best characterized of these are the11

three species that cause disease in humans: Y. enterocolitica and Y.12

pseudotuberculosisarezoonoticpathogensthatcauseself-limitinggastroenteritis13

inhumans,whereasY.pestis is a rodent and fleapathogen that is occasionally14

transmittedtohumansandisthecausativeagentofplague27.Theremaining1515

knownspeciesofthisgenusarecommonlyfoundinsoilandwaterenvironments16

andaregenerally considered tobenon-pathogenic tohumans, thoughYersinia17

ruckeri is thecausativeagentof redmouthdisease insalmonids21andYersinia18

entomophoga has insecticidal activity24. Recent phylogenomic analysis of the19

entire Yersinia genus allowed for an accurate, whole genome SNP-based,20

assessmentof itspopulationstructure8,showingtherewere14distinctspecies21

complexeswithin thegenus.This robust sequencedbased taxonomydisagreed22

with the standing taxonomic description of the genus, based largely on23

biochemical differences. It also showed that the mammalian pathogenic Y.24

enterocoliticaspeciesandY.pseudotuberculosisspeciescomplex(alsocontaining25

7

Y.pestisandY.similis)fellatoppositeendsoftheYersiniaphylogenicspectrum1

(Box1)anddidnotbranchtogetheraswaspreviouslythought.2

Earlymicrobial population genetics studies showed that Y.pestis is a recently3

emergedcloneof theenteropathogenY.pseudotuberculosis,yethasamarkedly4

different lifestyle and causes amuchmore acute disease28,29. The evolutionary5

narrative tells how Y.pestis evolved from the gastrointestinal pathogen, in an6

evolutionaryblinkof aneye (estimated tobebetween2,000and10,000years7

ago28,30) through the processes of gene gain, gene loss and genome8

rearrangements. This finding was further explored with early microbial9

comparativegenomicsstudies.TheserevealedthatY.pestishademergedfromY.10

pseudotuberculosis through amixture of both gene gain events, in the form of11

acquisition of species-specific plasmids, bacteriophage, integrons and genomic12

islands, as well as multiple complete or partial gene loss events leading to a13

reduction in genomic flexibility and metabolic streamlining. All of this is14

associatedwith its change in lifestyleandultimateniche restriction10,31–33.The15

relationship of Y.enterocolitica to the Y.pseudotuberculosis—Y.pestis complex16

was until recently poorly defined. In 2006, a comparison of the available17

genomesfromallthreespeciessuggestedthattheysharedadistantbutcommon18

pathogenic ancestor which had acquired key pathogenicity determinants19

including theessentialvirulenceplasmidpYV,encodingYsc theprototype type20

III secretion system (T3SS), before splitting into distinct lineages9,27. However,21

the availability of just a single Y.enterocolitica genome sequence at this time22

limited the interpretations that could be made. Further genomes were23

subsequently published for Y. enterocolitica in 2011 and 201334–37, as well as24

singular genomes for type strains of environmental species in 201038 but our25

8

understandingof theevolutionof thiswholegenusandwhereY.enterocolitica1

fittedintotheevolutionaryscaleofYersiniaspecieswasstilllimitedbythelack2

of comprehensive whole genome data. Nonetheless, even with this limited3

genomic data, a combination of gene gain and gene loss in the three human4

pathogenic Yersinia species was evident and highlighted the complexity of5

evolutionofmammalianpathogenicityinthesebacteria,showingthatpathogens6

canevolvebyaprocessof“addDNA,stir,andreduce”27.7

The study of the evolution of pathogenesis in the genus also suggested an8

inexorably distinct relationship between loss ofmetabolic function by genome9

decay, reduction in ecological flexibility, and the evolution towards niche-10

restricted highly pathogenic lineages27. As population genomic studies have11

becomeanestablishedtool inmicrobiology,thelevelofresolutionatwhichwe12

can study these evolutionary events has improved considerably. By genome13

sequencing across entire bacterial genera it is possible to determine the key14

acquisitionand lossevents in theevolutionofbacterialpathogenesis. It isalso15

possibletoshednewlightontheevolutionaryforcesandpatternsunderpinning16

theseprocesses,andidentifytheircommonfeaturesacrossbacterialgenera.17

18

Theroleofgenegain19

The evolutionarypath fromenvironmental or commensal bacterium tohuman20

pathogenhaslongbeenassociatedwiththegainofmobilegeneticelementsvia21

horizontal gene transfer, through phage transduction, plasmid uptake, natural22

transformationandDNAconjugation.IntheYersiniagenusthereseemstohave23

been‘footholdmoments’characterizedbyacquisitionsthat,lookingbackacross24

the genus phylogeny appear as pivotal steps in the emergence of mammalian25

9

pathogenic lineages. The most important of these seems to have been the1

acquisitionof the familyofpYVvirulenceplasmidsencoding theYscprototype2

T3SS8,12,13,39.TheYscT3SSdeliversYopeffectorproteinsdirectlyintohostcells3

uponcontact, resulting in thecessationofactinpolymerization forendocytosis4

(which promotes bacterial uptake) and in the suppression of host-cell5

transcription40. Following contactwith hostmacrophages, the activities of Yop6

effectorssilence the innate immuneresponse, resulting in thedown-regulation7

of pro-inflammatory cytokine production and allowing proliferation of the8

infection41–43,aprocesstermedthe“Yersiniadeadlykiss”.9

The advantage of sequencing broadly across a genus and deep within each10

speciesisthattheoriginsanddistributionofallgenesdescribedasYersiniaspp.11

virulence factors, orotherwise, in thepublished literature canbeascertained8.12

Suchananalysis confirmed thatpYVwasoneof only twovirulence-associated13

genetic loci thatwereuniquelypresent inall threeYersinia lineagescommonly14

associatedwithmammalian disease (pYV is absent fromYersinia species non-15

pathogenictomammals).Theotherwasthechromosomallyencodedattachment16

and invasion locus ail which has a role in the attachment to, and invasion of,17

mammalian epithelial cells as well as in resistance to killing by the host18

complementsystem44–46.ThepivotalroleofpYVintheemergenceofmammalian19

pathogenesis is highlighted by the revelation that, contrary to accepted20

wisdom9,27,47, closely related versions of this plasmid were acquired21

independentlyonat least threeoccasions in theYersiniagenus; twicebytheY.22

enterocoliticaandoncebytheY.pseudotuberculosis—Y.pestislineage8,andthat23

Y. enterocolitica did not share a common pathogenic ancestor with Y.24

pseudotuberculosisaspreviouslythought8.Assuch,multipleindependentailand25

10

pYV acquisitions were key gene gain events that marked the parallel1

independentevolutionofmammalianpathogenesisintheYersiniagenus.Thisis2

surprising ifoneconsidersthe largenumberofvirulence-associatedgenesthat3

have been reported in Yersinia based on published classic bacterial genetics4

experiments.This includeskeyvirulence factorssuchas Invasin48, theFes/Fep5

ironacquisition system49, theTad type IVbpilus50 andMyf fimbriae51, and the6

Ystheatstabletoxin52,allofwhicharewidelydistributedacrosspathogenicand7

non-pathogenicmembersofthegenus.Thiscouldbeexplainedthroughtheeco-8

evo perspective whereby the environmental yersiniaemay have acquired and9

maintained some virulence factors to combat predatory protozoa, nematodes,10

predatorybacteria,andbacteriophageoreventocoloniseplants.Thishighlights11

the benefits of studying the accessory gene pool of pathogenic bacteria in the12

widercontextofthegeneratheybelongto,astrategythathasyettobewidely13

applied to other enteric pathogens. Indeed in order to confirm the eco-evo14

hypothesisthereisalsoabenefitandneedtostudytheroleofvirulencegenesin15

thesenon-pathogenicisolatestoallowustofullydeterminethetrueecologyof16

bacterialspecies.17

In addition to the crucial gains of the pYV plasmid and ail locus in all three18

human pathogenic Yersinia spp., a number of other acquired loci have been19

maintained by clonal expansion in individual Yersinia species complexes and20

phylogroups (PG). Perhaps the most striking and formative of these is the21

acquisition of the pFra (pMT1) and pPla (pPCP1) plasmids of Y.pestis, which22

encodethemurinetoxinYmtandtheF1capsularproteinandtheplasminogen23

activatorPla.Theacquisitionofthesetwoplasmidsisasignificantevent inthe24

movetowardsefficientinsectvector-mammaliantransmissionofY.pestis,53and25

11

Plahasrecentlybeenshowntobepivotalforcausingthefulminantpneumonic1

infection unique to Y.pestis within the Yersinia genus54. Also in this group of2

acquired elements is the High Pathogenicity Island (HPI). HPI is a large3

integrative-conjugative element capable of excision and transfer via the use of4

selfencodedintegraseandexcisionfactors55.AssuchtheHPIisoftenfoundina5

rangeofbacterialgeneraincludingextra-intestinalpathogenicE.coli56.TheHPI6

encodes the siderophore yersiniabactin that sequesters iron in the host56,57,7

imports zinc into bacterial cells58, and assists in reducing the respiratory8

oxidativeburstresponseofimmunecells59.WithinYersiniaspp.,HPIcontaining9

a fully functioning yersiniabactin is found only in strains belonging to the Y.10

pseudotuberculosis—Y. pestis species complex, where its presence is isolate11

dependant, and in the highly virulent phylogroup 2 lineage of the Y.12

enterocoliticaspeciescomplex,whereitisuniformlypresent.Thekeyremaining13

gene acquisition events that have occurred in the pathogenicmembers of the14

genusinvolvetheYsaT3SSbyphylogroup2Y.enterocolitica,andHmsbytheY.15

pseudotuberculosis—Y. pestis species complex. The Ysa T3SS is located in a16

region of the genome known as the plasticity zone60, first identified in the17

archetypal phylogroup 2 strain 80819. The plasticity zone is a region of theY.18

enterocoliticagenomethatshowssignsofmultipleindependent,andphylogroup19

specificacquisitionsanddeletions9.YsaT3SShasbeenshowntosecretealarge20

numberof chromosomallyencodedT3SSeffectorproteins61aswellas theYop21

T3SSeffectorproteinsoftheYscsystemcarriedonpYV62.Althoughmutagenesis22

studies suggest ysa plays only a minor role in mammalian pathogenesis61,23

consistent with the eco-evo perspective, ysa mutants were shown to be24

attenuated in their ability to survive inside cultured Drosophilamelanogaster25

12

cells suggesting ysa is more important outside of the mammalian host for1

phylogroup2Y.enterocolitica63. TheHms locus is locatedon thechromosome2

andencodesaheminstoragelocusthatisuniquetotheY.pseudotuberculosis-Y.3

pestis lineage and is responsible for its ability to form pigmented colonies on4

congo-redagar64.HmsisimportantforY.pestispathogenesisasitisresponsible5

for formation of biofilms, required for the blockage of the flea foregut, which6

facilitates its subsequent transmission to mammals through reflux of infected7

bloodfromtheinfectedfeedingfleabackontothebitesite65.8

9

Regulatorycontrol10

Although theacquisitionofmobile genetic elementshasbeenakeyprocess in11

the emergence of Yersinia spp. pathogenesis, loci aremore likely to be stably12

maintainedinapopulationifthereisaminimalfitnesscosttotheirintegration13

intothegenomeofthehostbacterium,orahighselectivepressureforthemto14

be maintained. To achieve this, many acquired elements fall under strict15

transcriptional and/or translational control to minimise the fitness costs16

associatedwith theiracquisition.Across theEnterobacteriaceaemanyacquired17

genetic elements are regulated by H-NS, a nucleoid associated, histone-like18

proteinthatbindstoA-TrichregionsofDNAsilencingtranscriptionalactivity66.19

Onceunder thestrictnegativecontrolofH-NS, theacquiredelementsare then20

integrated into existing transcriptional activating regulonswithin the bacterial21

cell. These function in an antagonistic fashionwithH-NS to tightly control the22

transcriptional expression of the new genes. In the case of Yersinia spp., the23

chromosomal rovA/ymoA regulonhasbeen suggested to fulfil this antagonistic24

role to H-NS8,67. The rovA gene is universally present across the genus8 and25

13

encodesamemberoftheMarR/SlyAfamilyofglobaltranscriptionalregulators67.1

It is known to antagoniseH-NS silencing of the invasin protein gene, invA, the2

product ofwhich is involved in the early stages ofYersiniaspp. attachment to3

cellsand internalizationduring infection.ThebindingofRovAto thepromoter4

regionmaskstheH-NSbindingsite,triggeringtranscription68.GenesrovA/ymoA5

are thought to encode a promiscuous ancestral regulon into which the6

expressionofacquiredgeneswere incorporatedmultiple times in thedifferent7

lineages (FIG 1). This is thought to explainwhy RovAregulates different gene8

sets inY.enterocoliticaandY.pseudotuberculosis/pestis67,69:RovAwasshownto9

transcriptionallyactivate73genes inY.pestis and63genes inY.enterocolitica,10

but only three genes were common to both species67. The majority of RovA11

controlled genes are lineage-specific virulence factors such as Invasin or Myf12

fimbriae, or lineage specific prophage or metabolic genes67. The rovA/ymoA13

regulon also controls the expression of genes encoded on the pYV plasmid,14

highlightingitsimportanceasaglobalregulatorofvirulence.15

The expression of the Yop proteins encoded on pYV is down-regulated at a16

transcriptionallevelbyLcrF(anorthologofHN-S,encodedonthepYVplasmid),17

inresponsetothemammalianenvironment.Recentworkhasidentifiedthatthis18

regulation is controlled at a hierarchical level by YmoA, which functions as a19

thermo-sensing switch to turn off lcrF transcription, and therefore Yop20

translation and secretion at low temperatures70, and by Hfq and other small21

regulatoryRNAsconserved in theYersiniagenus71.This thermo-sensingswitch22

is a tandem process which relies on weak binding of YmoA directly to the23

promoterregionoflcrFatlowtemperaturesandtheformationofadoublestem24

loop in the intergenic region resulting in stearichindranceofRNApolymerase25

14

binding and formation of transcripts70,72. Overall the importance of the1

RovA/YmoAsystemfortheevolutionofpathogenesisinYersiniaisevidentfrom2

these data: At temperatures below 37OC virulence factors, essential for3

mammalianpathogenesis,arenotexpressedbecauseRovA/YmoAisinactiveand4

theyarerepressedbyH-NS.Howeverflagellabiosynthesisgenes(inthecaseof5

the enteropathogenic species) or insect transmission loci such as hms (in the6

case of Y.pestis)are expressed at high levels73,74. Upon a shift to mammalian7

bodytemperaturesessentialacquiredvirulencefactorssuchasthoseonpYV,ail8

and invA are activated by RovA/YmoA.Other environmental triggers are also9

knowntoinfluencethisregulatorycascadesuchaslevelsofexogenouscalcium10

ions75 or levels of exogenous magnesium ions via the PhoPQ two component11

regulatory system76. Indeed PhoPQ has been shown to have an overarching12

controlonlevelsofRovAandassuchakeyroleinvirulencephenotypesinall313

humanpathogenicspecies77–79.14

The presence of an ancestral promiscuous regulatory system controlling15

expressionofacquiredgenesinvolvedinvirulencedrawsstrikingparallelswith16

Salmonellaspp.Thekeyacquisitioneventintheevolutionofpathogenesisinthe17

Salmonella genus was Salmonella pathogenicity island-1 (SPI-1). This18

pathogenicity island encodes aT3SS and its cognate secreted effectorproteins19

thatremodeltheactincytoskeletonofinfectedcellsandforceuptakeofattached20

bacteria into endocytic vacuoles. As with pYV, the acquisition of SPI-1 was a21

landmarkevent intheevolutionof theSalmonellagenus,beingpresent inboth22

species: S. enterica and Salmonella bongori80. SPI-1 is under negative23

transcriptionalcontrolbyH-NS.SimilartoYersinia,H-NS-mediatedrepressionof24

SPI-1 functions is alleviated by the concerted action of a number of25

15

transcriptional regulators including HilD/HilA, SlyA which is an ortholog of1

RovA,andthetwo-componentregulatorysystemPhoPQ81.Recentworkshowed2

thatSPI-1wasreadilylost inSalmonellaH-NSnullmutants,ShowingthatH-NS3

silencingofSPI-1waslikelytohavebeenanessential factor inSPI-1becoming4

fixed inallSalmonellaspp.andsotheevolutionofpathogenesis inmembersof5

thisgenustoo82.6

Therefore it seems thatanemerging theme in theevolutionofpathogenesis in7

entericbacteriaisnotonlythepivotalacquisitionofoneortwokeygeneticloci,8

butalsotheco-ordinatedtranscriptionalcontrolofthoseacquiredlocibyH-NS9

andotherspecies-specificpromiscuousglobalregulators.10

11

Roleofgeneloss12

Althoughtheimportanceofgenegainintheevolutionofbacterialpathogenicity13

is intuitiveandwelldocumented, theroleofgene lossandhostadaptationhas14

been shown to be central in the emergence of bacterial pathogens83. The15

majority ofwork looking at the evolution of pathogenesis inYersinia spp. has16

focusedontheevolutionaryeventsthatledtotheemergenceofY.pestisfromY.17

pseudotuberculosis28. Although there were important acquisition events, the18

most striking observation of early comparative genomic analyses was the19

functional loss of a large number of genes associated with virulence and20

metabolisminY.pestis10.Thisfunctionallossappliedtoabout10%ofthegenes21

inthegenome,aconsiderablylargerproportionofthegenomecomparedtogene22

gainevents.Thegeneattritionwaslargelyattributabletotheexpansionoffour23

majorclassesofISelements(IS1541,IS100,IS285,IS1661)leadingtoinsertional24

gene inactivation. Other processes of gene loss included deletion of genes and25

16

lociandSNP-basedpseudogeneformation32,33,84,85.Manyofthegenesthatwere1

functionally lost encode virulence factors such as invasin and YadA, and2

metabolic loci involved in motility, dicarboxylic amino-acid metabolism, and3

uracil transport10,27. These are required for successful colonization of the4

mammalian gastrointestinal tract, a niche no longer frequently utilised by Y.5

pestis as it emerged from the intestinal pathogenic Y. pseudotuberculosis to6

colonize the mid-gut of fleas and, in mammals, to become a more systemic7

pathogeninvadingthelymphaticsystem27.8

The importance of gene loss in the evolution of Y. pestis has recently been9

exemplified by studies investigating key mutations in Y. pestis that mark its10

evolutionary adaptation to flea-borne transmission in mammals. Using a11

combinationofcomparativegenomicsandclassicalbacterialgenetics,threekey12

loss-of-functionmutationswerefound,allofwhichincreasedbiofilmformation13

in the flea foregut in a cyclic-di-GMP dependant manner6. The key mutations14

were in two genes encoding EAL-domain phosphodiesterases (PDE), and the15

rcsA gene that is a component of the rcs regulatory network. The rcs system16

transcriptionally represses the hms locus and biofilm formation. PDE are17

enzymes that degrade cyclic-di-GMP, a bacterial signalling molecule that18

enhancestranscriptionofhmsandbiofilmformation6.Thefunctionallossofthe19

PDE enzymes and the regulator repressing transcription in a Y.20

pseudotuberculosis-ancestor led to the emergence of a lineage with enhanced21

abilitytoformbiofilmsintheforegutoffleasandaresultingshiftinpathogenic22

lifestyleofY.pestis6.TherelativelyrecentemergenceofY.pestis28 is consistent23

with the SNP-based pseudogenes,which have swept to fixation through theY.24

pestis population but have not yet been fully deleted30,86. Also in Y.pestis is a25

17

mutation in the ureD gene that leads to functional loss of urease. In Y.1

pseudotuberculosisithasbeenshownthatthefunctionalureDencodingureaseis2

associated with significant oral toxicity in the flea87, hence the ureDmutation3

may play a contributory role in the emergence ofY.pestis6,87(Fig 2). However,4

perhaps significantly the ureD mutation appears to be a reversible phase-5

variable mutation and maybe a contingency gene required in the mammalian6

hostorsoildwellingstage, rather than the insectvector,partof its lifecycle88.7

ThisprovidesY.pestistheflexibilityofretainingureaseexpression,asandwhen8

required.9

GenomicanalysisofY.enterocoliticashowedthatsimilargenelossandmetabolic10

restriction also mark the evolution of certain subtypes of this species8. As11

discussedaboveandshowninFigure3Y.enterocolitica canbesubdivided into12

the non-pathogenic phylogroup PG 1,which is an ancestral lineage, the highly13

pathogenicPG2,andPGs3-5whichshowlimitedpathogenicityinmousemodels14

butarethemostsuccessfullineagesintermsofdiseasecausationandaremost15

commonlyisolatedfromlivestockandhumanclinicalcases.PG6isararelineage16

whichhasonlyeverbeenisolatedfromwildhares89,90.Theevolutionarydescent17

from host-generalists (PGs 3-5) to the host-restricted PG 6, similar to the18

emergenceofY.pestis,wasaccompaniedinY.enterocoliticabytheexpansionofa19

single IS element, IS1667, as well as functional loss of a large number of20

metabolic genes. The impact of the functional loss of metabolic loci is21

particularly pronounced in PG 6 and is thought to reflect its extreme niche22

restrictiontohares.InterestinglythelistofmetabolicpathwaysinactivatedinPG23

6issimilarinparttothoselostinY.pestis(cobalaminbiosynthesis,tetrathionate24

respirationandthehydrogenase-4operonresponsible fortheuseofmolecular25

18

hydrogen inanaerobicrespiration).Thesemutationalobservationsaredirectly1

supportedbyhighthroughputmetabolicphenotypingofPG68.2

Lookingat the functions thatwere lost asdifferent lineagesofY.enterocolitica3

divergedandadaptedtonewniches,oneoftheearliestevents is likelytohave4

beenthedecayoftheYersiniagenusT3SS,YGT.YGTisanancestralT3SSpresent5

across thewholegenusand isdistinct fromtheYscandYsaT3SSsacquired in6

pathogenic lineages. YGT has yet to be fully characterized but it shares a high7

levelofsequenceidentitywiththeT3SSencodedontheSPI-2genomicislandin8

Salmonella enterica, which was a key acquisition required for intracellular9

survival91. The YGT present in non-pathogenic PG 1 Y.enterocolitica seems to10

containafullcomplementofeffectorproteinencodinggenescomparedwiththe11

SPI-2 T3SS8. In the low-pathogenic PGs 3-6 the YGT seems to have been12

inactivated either by single base pair mutations or deletion of the secretion13

apparatusgenes,whereasalmostalltracesoftheYGThavebeendeletedinthe14

highlypathogenicPG2isolates8.Asanexampleofhowdynamictheevolutionof15

pathogenesis is through gene gain and loss, the loss of YGT in the high-16

pathogenicPG2Y.enterocoliticais,conceptuallyatleast,offsetbythegainofthe17

Ysa T3SS60. A full functional characterisation of YGT is now required to18

determine if the selective maintenance of YGT in PG 1 Y. enterocolitica is a19

classical example of eco-evo principal, where the T3SS could be conferring a20

protectivebenefitinanon-mammalianhostenvironment.21

IS mediated gene inactivation is also evident in the low-pathogenic Y.22

enterocolitica PG 3 lineage (comprising the classically entitled serotype O:323

strains)92,93. Molecular analysis revealed a IS1667 had inserted into the invA24

invasin promoter region, resulting in the formation of an additional RovA25

19

binding site and a concomitant increase in expression of invA94. Comparative1

analysiswith theother low-pathogenicphylogroupsofY.enterocolitica suggest2

this is theprimaryevent leading to the enhancedvirulencepropertiesofPG33

isolatescomparedtoPGs2,4,and593.Asaresultoftheincreasedexpressionof4

invasionPG3 isolates can coloniseporcine tissuesmore effectively thanother5

phylogroups, and as result PG 3 have become the predominant isolates taken6

fromhumanclinical cases inEuropeover thepast10yearsandalsodominate7

veterinaryepidemiologicalsurveys95.8

9

GenelossinotherEnterobacteriaceae10

FunctionalgenelossinmetaboliclocihasbeenakeyeventintheYersiniagenus11

that mediated the transition from ubiquitous host-generalist lineage to niche12

restrictedandhost-adaptedpathogenonatleasttwooccasions.Onceagainthis13

observation has also beenmade in Salmonella species. S.Typhi96 showsmany14

similaritieswithY.pestis,it isniche restricted (in this case to thehumanhost)15

andcausesanacutesystemicdiseaseknownastyphoid.SimilarlyS.Gallinarum16

andS.Pullorumalsocauseatyphoid-likediseasebutarerestrictedtogalliforme17

birds97, whereas S.Typhimurium is a host-generalist causing an inflammatory18

intestinal infection of mammals98. Comparative genomic studies of these19

salmonellaehave shown that functional gene loss in loci involved in anaerobic20

metabolism (more specifically functional loss in the cob/pdu and ttr operons)21

wasamarkerofthetransitionfromintestinalpathogen,likeS.Typhimurium,to22

invasivepathogenssuchasS.Typhi,S.GallinarumandS.Pullorum99.23

InS.Typhimuriumunderanaerobicconditions,thecobgenesencodetheability24

tosynthesiscobalamin(orvitaminB12).Cobalaminisrequiredasacofactorfor25

20

several enzymes including the first enzyme in the 1, 2-propanediol utilisation1

pathway (encoded by pdu operon). 1,2-Propanediol is a by-product of2

fermentativegrowthonrhamnoseandfucose100,commonconstituentsofplant3

cellwallsandintestinalepithelialcells(andthereforethegut)101.Theanaerobic4

degradation of 1,2-propanediol requires tetrathionate for use as a terminal5

electron acceptor, facilitated by the products of the ttr genes102. Unlike 1,2-6

propanediol,tetrathionatewasonlypreviouslyknowntobepresentinsoilsnot7

inthemammaliangut.However,ithasbeenrecentlyshownthattetrathionateis8

producednaturallyduringaninflammatoryresponsetoaSalmonellainfection.It9

isundersuchconditionsthatthecob,pduandttrfunctionscombinetoprovidea10

competitivegrowthadvantageforS.Typhimurium,byallowingittooutgrowthe11

natural and largely fermentative gut microbiota by using naturally occurring12

carbon sources such as 1,2-propanediol that are not readily fermented103,10413

(FIG. 4). As the invasive typhoidal Salmonella have evolved away from an14

intestinallifestyle,functionsincob/pdu/ttroperonshavebeensequentiallylost15

andsorepresentmarkersof theirchange innicheandamovementaway from16

causingintestinaldiseasetoamoresystemicinfectioncycle105.17

Althoughthecob/pduandttr lociarepresentand intact inY.enterocoliticaand18

inthemajorityofenvironmentalYersiniaspecies106,theyareabsentfromtheY.19

pseudotuberculosis—Y. pestis complex. As mentioned above, tetrathionate is20

produced in the vertebrate inflamed gut following the host’s response to S.21

Typhimuriuminfection,wherebytheSPI-1and-2encodedT3SSareessentialfor22

stimulatingthisinflammatoryresponse.StudiesofY.enterocoliticapathogenesis23

have shown that infections are characterized by inflammation of the gut107.24

Moreover,inflammationappearstorequirepYV108.Itispossiblethatbyinducing25

21

a pYV-associated inflammatory response during intestinal infection109, and by1

expression of the cob/pdu and ttr loci, Y. enterocolitica is able, like S.2

Typhimurium,togainametabolicadvantageovertheresidentgutmicrobiotaby3

using tetrathionate and naturally occurring carbon sources to respire under4

theseconditions.Thefactthatthecob/pduandttr lociare lostcompletely inY.5

pseudotuberculosis–Y.pestismayexplainwhythesepathogensareincapableof6

causing the inflammatory intestinal infection observed in Y. enterocolitica,7

despitecontainingpYV.8

9

Summaryandconcludingremarks10

For decades the pathogenic yersiniae have been at the vanguard of11

understanding bacterial pathogenesis. Examples include the role of T3SSs42,11012

and understanding the molecular mechanisms by which intestinal pathogens13

invadetheintestinalepithelium44.WorkonthepathogenicYersiniaspeciesalso14

drovetheunderstandingoftheimportanceofplasmidsinconferringvirulenceto15

pathogenicbacteria12,39.Theyersiniaearealsoattheforefrontindevelopingour16

understanding of the evolution of mammalian pathogenesis. Furthermore,17

Yersiniaspp.genomicshasalsobeenatthefrontlineofpalaeomicrobiology,with18

the near complete reconstruction and interpretation of the Black Death and19

JustinianplagueY.pestisgenomesfromancientDNAstudiesonhistoricaldisease20

episodes86,111. Additionally the microevolution of Y. pestis is beginning to be21

unravelledandthedistinctionbetweenancestralandmodernY.pestishasbeen22

made 112.Andvery recent advanceshave identified thatY.pestiswas ahuman23

pathogen of bronze-age humans, but had not yet acquired Ymt meaning it24

evolvedtheabilitytoinfectfleasafterbecomingahumanpathogen113.25

22

The position of Yersinia as a model genus for pathogenesis has been further1

cementedasitisthefirstmultispeciesbacterialgenusforwhichallspecieshave2

beensequencedandplacedintophylogenomiccontext8.Bytakingthisapproach3

coupledwithhighthroughputmetabolicanalysesusingphenotypemicroarrays4

it was possible to trace the evolutionary origins of the species, contextualise5

themandrevealtheparallelevolutionarypathsofvirulenceinhumans8(FIG.5).6

The genus Yersinia evolved from environmental ancestors. The most outlying7

speciesarethefishpathogenicY.ruckeri(SC2),andnewlyclassifiedlineagesY.8

nurmii andY.entomophaga(SC 3). Previously, it had been debatedwhetherY.9

ruckeri was truly part of the genus Yersinia21, however this has now been10

strengthened by the recognition of this novel lineage. The human pathogenic11

species clustersemerged independently following the common themesof ‘add,12

stir,andreduce’.Y.pestisacquireditsuniquevirulenceplasmids,anexpansionof13

severalISelementsledtowidespreadgenomerearrangements,andconsiderable14

gene loss can be observed. However, in the Y. enterocolitica lineages, these15

themes are weighted differently in the various phylogroups. PG 2 added the16

high-pathogenicityisland,andtype2and3secretionssystems,andontheother17

hand lost the second flagella cluster and the genus T3SS YGT. PGs 3-6 show18

genomerearrangements following theexpansionof IS1667.Thesephylogroups19

alsoshowgenerallossofmetabolicflexibility,andinthecaseofPG6anextreme20

lossofmetaboliccapability.21

Comparative analysis of complete genome sequence data sets of all22

representative yersiniae species coupled with the eco-evo perspective of23

bacteriahasprovidedagreaterunderstandingofthekeyevolutionarystepsthat24

led to the emergence of successful mammalian pathogens. We need to move25

23

away from our biased human-centric view of bacteria and consider more the1

environmentalcontext,habitatandnichesoftheseorganisms.2

Finally, theYersiniagenus data shows that a genus-wide sequencing approach3

leads tonew informationon thedistributionofvirulence-associatedgenesand4

onwhichof these genesaregenuinevirulence factors that areappropriate for5

noveldiagnosticandpathogen-targetingresearch.However,evenwiththelarge6

numberofYersiniaspp. isolates sequenced todate, the origin and reservoir of7

mobilegenetic elements, suchaspYV, remainobscure. Itmaybe thatwehave8

onlysequencedthetipofthebacterialicebergandthatfuturelargegenomeand9

microbiome studies coupledwithhigh throughputphenotypicmethodswill be10

even more revealing. This will provide a web of bacterial life, the inter-11

relatednessofbacteria,nodoubtblurringspeciesboundaries.Thegenomedata12

together with the eco-evo perspective will form the basis of how and why13

bacteriaevolvetobemorepathogenicwiththepotentialtoavertfuturethreats14

posedbyoldadversariessuchastheemergenceofantimicrobialresistance.15

16

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82. Ali,S.S.etal.SilencingbyH-NSpotentiatedtheevolutionofSalmonella.21PLoSPathog.10,e1004500(2014).22

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84. Morelli,G.etal.Yersiniapestisgenomesequencingidentifiespatternsof28globalphylogeneticdiversity.Nat.Genet.42,1140-3(2010).29

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88. Sebbane,F.,Devalckenaere,A.,Foulon,J.,Carniel,E.&Simonet,M.4SilencingandreactivationofureaseinYersiniapestisisdeterminedbyone5GresidueataspecificpositionintheureDgene.Infect.Immun.69,170–6176(2001).7

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93. Schaake,J.etal.HumanandanimalisolatesofYersiniaenterocoliticashow23significantserotype-specificcolonizationandhost-specificimmune24defenseproperties.Infect.Immun.81,4013–4025(2013).25

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96. Parkhill,J.etal.Completegenomesequenceofamultipledrugresistant35SalmonellaentericaserovarTyphiCT18.Nature413,848–852(2001).36

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98. McClelland,M.etal.CompletegenomesequenceofSalmonellaenterica1serovarTyphimuriumLT2.Nature413,852–856(2001).2

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100. Badia,J.,Ros,J.&Aguilar,J.Fermentationmechanismoffucoseand8rhamnoseinSalmonellaTyphimuriumandKlebsiellapneumoniae.J.9Bacteriol.161,435–437(1985).10

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101. Bry,L.,Falk,P.G.,Midtvedt,T.&Gordon,J.I.Amodelofhost-microbial12interactionsinanopenmammalianecosystem.Science273,1380–138313(1996).14

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102. Price-Carter,M.,Tingey,J.,Bobik,T.A.&Roth,J.R.Thealternativeelectron16acceptortetrathionatesupportsB12-dependentanaerobicgrowthof17Salmonellaentericaserovartyphimuriumonethanolamineor1,2-18propanediol.J.Bacteriol.183,2463–2475(2001).19

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Keyaccountoftheroleofcob/pdu/ttrininflammationandintestinal23colonisationbySalmonella24

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109. Buret,A.,O’Loughlin,E.V,Curtis,G.H.&Gall,D.G.EffectofacuteYersinia3enterocoliticainfectiononsmallintestinalultrastructure.Gastroenterology498,1401–1407(1990).5

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110. Tardy,F.etal.YersiniaenterocoliticatypeIIIsecretion-translocation7system:channelformationbysecretedYops.EmboJ.18,6793–67998(1999).9

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111. Wagner,D.M.etal.YersiniapestisandtheplagueofJustinian541-543AD:11agenomicanalysis.Lancet.Infect.Dis.14,319–326(2014).12

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112. Cui,Y.etal.Historicalvariationsinmutationrateinanepidemicpathogen,14Yersiniapestis.Proc.Natl.Acad.Sci.U.S.A.110,577–582(2013).15

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ago.Cell.163,1-12(2015).1819

114. Hall,M.etal.Useofwhole-genusgenomesequencedatatodevelopa20multilocussequencetypingtoolthataccuratelyidentifiesYersiniaisolates21tothespeciesandsubspecieslevels.J.Clin.Microbiol.53,35–42(2015).22

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correlates.MicrobesInfect.1,323–333(1999).2526

116. Barnes,P.D.,Bergman,M.A.,Mecsas,J.&Isberg,R.R.Yersinia27pseudotuberculosisdisseminatesdirectlyfromareplicatingbacterialpool28intheintestine.J.Exp.Med.203,1591–1601(2006).29

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117. Bottone,E.J.Yersiniaenterocolitica:thecharismacontinues.Clin.31Microbiol.Rev.10,257–276(1997).32

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118. Sebbane,F.,Jarrett,C.O.,Gardner,D.,Long,D.&Hinnebusch,B.J.Roleof34theYersiniapestisplasminogenactivatorintheincidenceofdistinct35septicemicandbubonicformsofflea-borneplague.Proc.Natl.Acad.Sci.U.36S.A.103,5526–5530(2006).37

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119. Sha,J.etal.CharacterizationofanF1deletionmutantofYersiniapestis39CO92,pathogenicroleofF1antigeninbubonicandpneumonicplague,and40evaluationofsensitivityandspecificityofF1antigencapture-based41dipsticks.J.Clin.Microbiol.49,1708–1715(2011).42

43

35

Box1:PhylogenomicsofYersiniaspecies:approachesandmajorfindings.1

Traditionally, bacteria are speciated using limited biochemical tests that are2

basedonthephenotypicexpressionornon-expressionofatrait.Thiscanleadto3

problems in standardising methods, and the potential to mutate the trait can4

impactonthephenotype.Genomicinformationislessambiguous,andvariations5

within genes under neutral selection can be used to infer evolutionary6

relationships,providinga robust framework that is independentofphenotypic7

variation. For the genusYersinia, 84 housekeeping geneswere identified, that8

showedbetween10-25%SNPdivergencebetweenY.pestisandY.enterocolitica8.9

These criteriameet those of housekeeping genes used for the design ofmulti-10

locussequencetyping(MLST)schemes114,sothattheaccumulationofvariation11

reflects the history of the genus, and amaximum likelihood phylogeny can be12

estimated. Furthermore, Bayesian analysis of the population structure was13

employed to identify defined species clusters (SC8). Thismeans that statistical14

probabilities are calculated to describe the variation hidden within the15

population,anddoesnotonlyconsiderthesequenceofthe84genesasawhole16

butasseparategeneentities.Someofthespeciesclustersthusdescribedcontain17

multiplespeciesasdefinedbyclassicaltechniques,suchasSCs13and14.Onthe18

other hand, other species likeY.frederiksenii, that showheterogeneity in their19

phenotype,aredistributedoverseveralSCs(SCs8,9,and14).Thishighlightsthe20

limitations of biochemical tests that fail to accurately reflect the relationships21

between thedifferentspecies.Theanalysisalsoemphasizes thepositionof the22

pathogenic species. The human pathogenic species are positioned at23

diametricallyoppositeendsofthetree,withthefishpathogenY.ruckeri(green)24

formingathirdoutlyingSC.Eventhoughnoclassicalrootofthetreeisgiven,Y.25

36

ruckerimightbeconsideredanoutgroupsinceitspositionwithinthegenushas1

previously been under debate. The position of Y.pseudotuberculosis—Y.pestis2

and Y. enterocolitica nevertheless supports independent evolution of their3

pathogenicpotential.4

It must be noted however, that speciation might involve more than purely5

genomic evidence, asY.pestis andY.pseudotuberculosis arehighly similarona6

genomic level, yetexhibitmarkedlydifferentmechanismsofdiseaseaswell as7

nichepreferenceandlifestyle.8

9

Box2:MechanismsofpathogenesisinYersiniaspp.10

Thethreehuman-pathogenicspeciesofYersiniashareakeygeneticcomponent11

intheirabilitytocausedisease,alargevirulenceplasmidnamedpYV(plasmidof12

Yersinia Virulence). Though commonly thought of as a single entity, pYV is13

actually highly variable between species and strains of species, containing14

different origin of replications and showing variable genetic architecture8,12.15

Whatiscommonamongstthemall isa largegenetic locusencodingfortheYsc16

type III secretion system (T3SS), responsible for the targeted delivery of Yop17

effectorproteinsintohostcells.TheYopsarethekeyvirulencefactorinallthree18

ofthepathogenicspecies.UponcontactwithhostmacrophagesYopsareinjected19

into the host cells, resulting in silencing of the pro-inflammatory cytokine20

responseandapoptoticdeathoftheinfectedmacrophages40.21

Y. enterocolitica/Y. pseudotuberculosis – are zoonotic infectious agents which22

causeinflammatoryintestinaldiseaseassociatedwithingestionviaconsumption23

ofundercookedorcontaminatedporkproductsandvegetables115.Thebacteria24

enter the small intestine where they attach to the intestinal epithelium.25

37

NumerousvirulencefactorshavebeenimplicatedinthisincludingtheMyf,YadA,1

and Ail adhesins as well as flagella18. The invasin protein then triggers2

internalisation of the bacteria, which translocate across the epithelium48.3

Bacteria replicating in the intestinal tract can also disseminate to lymphatic4

tissuesviainfectionandsilencingofphagocyticimmunecells116.Asaresult,this5

infection is often diagnosed as pseudo-appendicitis, and can lead to6

septicaemia117.7

Y. pestis –unlike Y. enterocolitica and Y. pseudotuberculosis, does not have an8

intestinal phase to its infection-cycle. Y. pestis host reservoir is rodent fleas,9

whereitformsabiofilmintheirforegut65.Whenthefleanexttakesabloodmeal10

thebiofilmisregurgitatedintothehostprey.Ifinjectedintothebloodstreamthe11

bacteriaimmediatelyencounterphagocyticimmunecellstriggeringactivationof12

the Ysc system and host-cell silencing, leading to septicaemic plague118. If13

injected into the extravascular part of the dermis, the bacteria travel to the14

lymphnodeswhereanacuteinfectionisestablishedleadingtotheformationof15

painful swellings at the lymph nodes, or buboes118. This is known as bubonic16

plague. If untreated the infection will spill back into the bloodstream with17

bacteriatravellingtothelungs,wherepneumonicplagueisestablished.Y.pestis18

hasacquiredadditionalplasmids,pPlaandpFrawhichplaypivotalroles inthe19

insect-infectionandsystemic-infectionphaseoftheorganism53,54,119.20

21

22

Figure 1: Diagrammatic comparison of the RovA/YmoA regulons of Y.23

enterocolitica,Y.pseudotuberculosis,andY.pestis. Genes activated by RovA are24

shown ingreen,andgenesrepressedbyRovAareshown inred.Genes inbold25

38

share common RovA regulatory patterns between Y.pseudotuberculosisand Y.1

pestis.Alsoshownare the lociactivatedandrepressedbyRovA inaconserved2

fashion. This shows that the key acquired pYV plasmid (denoted by the lcrF3

gene) is the only locus showing conserved RovA repression. The invasion4

encoding gene inv is also shown as activated by RovA in a conserved fashion,5

however the asterix indicates that this is not entirely conserved, as inv is a6

pseudogeneinY.pestis.Thelackofcommonactivatedandrepressedgenesinthe7

3speciesbyaconservedregulatorymechanismhighlightsthekeyroleplayedby8

theRovA/YmoAsystemsincontrollingexpressionoflineagedefiningloci.9

10

Figure 2: Gene loss in the emergence of Y. pestis from Y. pseudotuberculosis.11

Deletioneventsingenesencodingmotilityandthemetabolicabilitytocolonise12

themammalian intestinal tract in ancestralY.pseudotuberculosisoccurred as a13

resultofashiftawayfromthatenvironmenttowardsaninsect-vectorphaseof14

the life-cycle. As a result there was selection for deletion of genes encoding15

insect-toxins, including SNP-based reversible inactivation of the ureD gene,16

encodingurease,whichhashighoraltoxicityinfleas.Fortransmissionfromthe17

flea to occur Y. pestis must form biofilms in the flea foregut. SNP based18

pseudogene inactivation of rcsA resulted in increased expression of the hms19

operon which encodes biofilm formation. Similarly SNP based pseudogene20

inactivation occurred in a gene encoding a phosphodiesterase. The product of21

this gene degrades cyclic-di-GMP, a molecule that acts as a transcriptional22

activatorofhmsandbiofilmformation.23

24

39

Figure3:GenegainandlossintheY.enterocoliticaspeciescomplex.Thediagram1

showshow thespecies isorganised intophylogeneticallydistinctphylogroups,2

whichparallelLPSstructureandserotype.PG1ismostsimilartotheancestralY.3

enterocolitica. The emergenceof pathogenicity in the species ismarkedby the4

acquisitionofpYV,and inPG2theYsaT3SS.TheYGTT3SS is lost fromall the5

pathogenic phylogroups. The other significant event is the acquisition of6

serotypespecificLPSoperons ineachof thepathogenicphylogroups.PG3has7

becomethedominantisolatefrompigreservoirsandhumandiseasecases,and8

hasadeletionintheFlag-2secondaryflagellagenesaswellasaninsertioninthe9

invasinpromoter,whichincreasestranscriptionofthiskeyvirulencefactor.10

11

Figure4:EnteropathogenicSalmonellaandYersiniaspeciesusecob/pduandttr12

locitooutcompetenormalintestinalfloraduringmammalianinfection.Thecob13

genesencodecobalaminbiosynthesis.Cobalaminactivatesenzymesencodedby14

pduthatdegradeorethanolamine.Thisprocessrequirestheuseoftetrathionate15

asaterminalelectronacceptorinanaerobicrespiration,therespirationofwhich16

is encoded by the ttr genes. The enteropathogenic Yersinia and Salmonella17

species induce inflammationduring infection.This inflammation leads toover-18

productionofethanolaminebytheintestinalepithelium,whichcreatesahostile19

environment for gastrointestinal commensal bacteria, but a favorable growth20

environment for the enteropathogens. This leads to the enteropathogens21

outcompeting the normal flora and overgrowth and colonization by the22

pathogens.23

24

40

Figure 5: A diagrammatic overview of the evolution of pathogenesis in the1

Yersiniagenus,highlightingkeygenegainandgenelosseventsintheformation2

ofeachof thepathogenic lineages.AcquisitionofpYVoccurs independentlyon3

twooccasionswithinY.enterocoliticaPG2andPGs3-6,aswellasadditionallyin4

a Y. pseudotuberculosis ancestor. These two human-pathogenic lineages have5

emerged on independent occasions from environmental generalists following6

similar themes of gene gain, gene loss, metabolic reduction, and genome7

rearrangements.8

9

Glossary10

Compensatorymutation–Mutationsthatoccurtooffsetdetrimentalgenegain,11

loss,ormutationeventsinindependentpartsofthegenome.12

Conjugation -The transferofDNA–usuallyplasmids–betweenorganismsvia13

directcell-to-cellcontactorabridgebetweencells.14

Cyclic-di-GMP – Secondary messenger molecule used in bacterial signal15

transduction to modulate gene expression in response to environmental16

perturbations.17

Eco-evoperspective–Aperspectiveinwhichorganismsareevaluatedbroadlyin18

the light of evolution and ecology, rather than narrow constraints of their19

behaviourinthelaboratoryorinhumaninfection20

F1capsularprotein–ProteinantigenfoundonthesurfaceofpathogenicYersinia21

thoughttomodulatetargetingofbacteriatositesofinfection.22

Genomicislands-Largegeneticregionsthatarepartoftheaccessorygenepool.23

They form the horizontally acquired part of a genome encoding one of more24

functionalgroupsofgenes.TheyarefrequentlyassociatedwithtRNAgenesand25

41

are flanked by repeat structures, and contain mobility genes coding for1

integrasesortransposasesrequiredforchromosomalintegrationandexcision.2

Horizontal gene transfer - The transfer of DNA, frequently cassettes of genes,3

betweenorganisms.Thisprocessisincontrasttoverticalgenetransfer,whichis4

muchmorecommonandoccurswhengeneticmaterialispassedfromparentto5

offspringor,moregenerally,fromancestortodescendent.6

Integrons-Acassetteofgenesencodingasite-specificrecombinase/integrase,a7

recombinationrecognitionsite,andapromoter.Oftenfoundinconjunctionwith8

othergenessuchasantibioticresistancegenes.9

ISelement-Thesimplesttypeoftransposableelementinabacterium.Insertion10

Sequenceelementsencodeonlythegenerequiredforitsowntransposition,and11

isflankedbyrepeats.12

LEEpathogenicityisland–MobilegeneticelementencodingforaT3SSfoundin13

enteropathogenicandenterohaemmorhagicE.coli.Injectseffectormoleculeinto14

hostintestinalepithelialcellsresultinginactinrearrangementandformationof15

characteristicattachingandeffacinglesions.16

MarR/SlyA–familyoftranscriptionalregulatorsfoundinbacteria.Generallyact17

asactivatorsoftranscriptionbyalleviatingHN-Smediatedrepression.18

Natural transformation - Direct uptake of DNA from the environment and19

incorporationofthisgeneticmaterialintothechromosomebycompetentcells.20

Palaeomicrobiology – Study of ancient infectious disease outbreaks by21

recoveringnucleicacidsequencesfromancienthumanremains.22

Phenotypic microarray analysis – High-throughput, automated assays that23

determine the ability of bacterial strains tometabolisemetabolic substrates in24

parallel.25

42

Phylogenomic analysis – The use of whole genome sequences to create1

phylogenetic trees and infer high-resolution evolutionary patterns. This is in2

contrasttousingphylogeneticmarkerssuchas16S.3

Pla–PlasminogenactivatorproteinfoundinY.pestisandencodedonaY.pestis4

specificplasmid.Proteaserequiredforpneumonicinfection.5

Promiscuous(regulon)–Promiscuousregulatorsassumetranscriptionalcontrol6

of large numbers of genes (regulons) that do not come under fine-scale7

environmentalcontrol.ExamplesareHN-S,IHF,andFIS.8

Salmonids-Relatingtosalmonandtroutfish.9

Sideophore-Compoundenablinghighlyefficientcaptureofexogenousiron.10

Transduction - The transfer ofDNA – frequently cassettes of genes – between11

organismswiththehelpofphages.12

Two-componentregulatorysystem-Bacterialsensor-kinasesystemscomposed13

of an outer membrane sensor, which autophosphorylates in response to a14

specific environmental stimulus. This then leads to phosphorylation of a15

responseregulatorthatupanddownregulatesexpressionofoperons.16

Type III secretion system (T3SS) – A mechanism by which bacteria export17

proteins. Often responsible for the translocation of bacterial effector proteins18

from pathogenic or symbiotic bacteria directly into the cytoplasm of their19

eukaryotic host, where these proteins subvert eukaryotic-cell functions to the20

advantageofthebacterium.21

Verotoxin – Shiga-like toxin produced by verotoxigenic E. coli. AB5 toxin that22

targetscellsintherenalglomerulileadingtokidneydamage.23

Ymt–Yersiniamurinetoxin.Firstcharacterisedasadeterminantoflethalityin24

mice,nowknowntoplayacrucialroleintheabilityofY.pestistosurviveinfleas.25

43

Yops–YersiniaouterproteinsareasetofeffectorproteinssecretedbytheYsc1

T3SSfoundonthepYVplasmidinpathogenicYersiniaspp.Yopsareinjectedinto2

phagocyticcellswherethesilencetheproductionofpro-inflammatorycytokines3

andinduceapoptosisintheinfectedcell.4

5

44

AuthorBiographies1

AlanMcNallyisareaderinMicrobialGenomicsatNottinghamTrentUniversity.2

HestartedworkingonYersiniain2003asapost-docinthelabofDianneNewell3

at the veterinary laboratories agency in Surrey. Upon moving to NTU he4

continued his work on Yersinia,and now has his own group working on the5

evolution of pathogenesis in Yersinia, and the emergence of drug resistant6

lineagesofextra-intestinalpathogenicE.coli.7

NickThomsonisafacultymemberattheWellcomeTrustSangerInstitute.Heis8

amicrobiologistandbioinformaticianandisinterestedinbacterialevolutionand9

spreadwitha focusonsexually transmittedanddiarrhoealdiseases.He isalso10

ProfessorofBacterialGenomicsandEvolutionattheLondonSchoolofHygiene11

andTropicalMedicine.12

Sandra Reuter is a postdoctoral research associate at the University of13

Cambridge. She undertook her PhD with Alan McNally and Nick Thomson on14

Yersinia whilst at Nottingham Trent University, and continued some of this15

researchattheWellcomeTrustSangerInstitute.Sheisnowworkingonhospital-16

acquired infections,mainlymethicillin-resistantStaphylococcusaureus (MRSA),17

andantimicrobialresistance.18

BrendanWrenisProfessorofMicrobialPathogenesisattheLondonSchoolof19

HygieneandTropicalMedicinesince1999.Hisresearchgroupexploitsarange20

ofpostgenomeresearchstrategiestogainacomprehensiveunderstandingof21

howpathogensfunctionandhowtheyinteractwiththeirrespectivehosts.22

Currentresearchfocusesonglycosylationinbacterialpathogensanddeveloping23

a“glycotoolbox”forglycoengineering,comparativephylogenomicsandthe24

evolutionofbacterialvirulenceandmechanismsofbacterialpathogenesis.25

Y.  enterocoli,ca  species  complex    

Pathogenicity   None   High   Low  

Phylogroup  

Serotypes  

4  3  2  1  

O:5,  O:6,  30,  O:7,  8,  O:18,  

O:46,  nontypeable  

O:8,  O:4,  O:13a,  13b,  O:18,  O:20,  O:

21  

O:9   O:5,  27  

5   6  

O:3   O:2,  3  

Ecological  distribu8on  

Rare  isolate  from  humans  

and  livestocks  

Ubiquitous   Most  common  pig  and  human  isolate  

Pigs  and  humans  

Pigs  caMle  and  sheep,  

rarely  humans  

Hares  

Gene    gain/loss  

Gain:  pYV,  Ysa,  O:8  Loss:  Flag2,  LPS  outer  core,  YGT,  metabolic  genes  

Loss:  Flag  2   Gain:  pYV  Loss:  YGT,  metabolic  genes  

Gain:  pYV  Loss:  YGT,  extreme  metabolic  reducUon  Loss:  Flag  2,  Invasin  

expression  

Cobalamine   Ethanolamine  

Pdu  

Tetrathionate  

Pro-­‐inflammatory  signals  

Increase  in  Compe;;ve  growth  

Genus Yersinia

SC 6/7 enterocolitica

SC 1 wautersii/similis/

pseudotuberculosis

PG 1

SC 10 rohdei

pathogenic precursor biofilm, O:1b/O:3

PG 2 PG 3-6 pestis

SC 14 massiliensis

SC 2 ruckeri

SC 3 nurmii/entomophaga

pYV HPI T2SS Ysa T3SS

Flag2 YGT

Expansion of IS1667

Genome rearrangement

Loss of metabolic flexibility

Flag2 pYV / pCD Hms HPI tcPAI ail

pMT / pFra

pPla/pPCP Expansion of IS1541, IS100, IS285, IS1661

Genome rearrangement

Gene loss

SC 13 mollaretii/bercovieri/ aleksiciae

SC 4 aldovae

SC 5 kristensenii

SC 8 frederiksenii

SC 9 frederiksenii 2

SC 12 pekkanenii

SC 11 intermedia

pYV tcPAI

ail