GI-type T4SS-mediated horizontal transfer of the 89K pathogenicity island in epidemic Streptococcus suis serotype 2.

1.4 MB·English

by  Li, Ming

MolecularMicrobiology(2011)79(6),1670–1683 (cid:2) doi:10.1111/j.1365-2958.2011.07553.x
Firstpublishedonline10February2011
GI-type T4SS-mediated horizontal transfer of the 89K
pathogenicity island in epidemic Streptococcussuis
serotype 2
mmi_75531670..1683
MingLi,1,2XiaodongShen,3JinghuaYan,1 S. suis 2 recipients, where it reintegrates site-
HuimingHan,1,4BeiwenZheng,1,4DiLiu,1 specificallyintothetargetsite.Theconjugaltransfer
HaoCheng,1,4YanZhao,2XiancaiRao,2 of 89K occurred via a GI type IV secretion system
ChangjunWang,5JiaqiTang,5***FuquanHu2**and (T4SS)encodedin89K,atafrequencyof10-6transcon-
GeorgeF.Gao1,4,6* jugants per donor. This is the first demonstration of
1CASKeyLaboratoryofPathogenicMicrobiologyand horizontaltransferofaGram-positivePAImediatedby
Immunology,InstituteofMicrobiology,ChineseAcademy aGI-typeT4SS.Weproposethatthesegeneticevents
ofSciences,Beijing100101,China. are important in the emergence, pathogenesis and
Departmentsof2Microbiologyand3Biochemistryand persistenceofepidemicS.suis2strains.
MolecularBiology,ThirdMilitaryMedicalUniversity,
Chongqing400038,China.
Introduction
4GraduateUniversity,ChineseAcademyofSciences,
Beijing100101,China. Streptococcus suis serotype 2 (S. suis 2) is a Gram-
5DepartmentofEpidemiology,ResearchInstitutefor positive,economicallyimportant,zoonoticpathogenthatis
MedicineofNanjingCommand,Nanjing210002,China. aleadingcauseofcontagiousporcinediseases,including
6BeijingInstitutesofLifeScience,ChineseAcademyof meningitis, arthritis, pneumonia, endocarditis and septi-
Sciences,Beijing100101,China. caemia(Maetal.,2009;Fengetal.,2010).Further,S.suis
2canalsobetransmittedtohumansbydirectcontactwith
infected swine, causing meningitis, permanent hearing
Summary
loss,septicshockandevendeath(Gottschalketal.,2007).
Pathogenicity islands (PAIs), a distinct type of Due to its high incidence, transmissibility, persistence in
genomicisland(GI),playimportantrolesintherapid the environment and its ability to occur in explosive epi-
adaptationandincreasedvirulenceofpathogens.89K demicform,S.suis2continuestobeaglobalpublichealth
is a newly identified PAI in epidemic Streptococcus concern.Aftertwoextensivehumanoutbreaksofstrepto-
suis isolates that are related to the two recent large- coccaltoxicshocksyndrome(STSS)causedbyS.suis2in
scaleoutbreaksofhumaninfectioninChina.However, Chinain1998and2005(Tangetal.,2006),S.suis2has
its mechanism of evolution and contribution to the beenrecognizedasare-emergingzoonoticpathogenand
epidemicspreadofS.suis2remainunknown.Inthis wasfoundtocharacteristicallyharbouracandidatepatho-
study,thepotentialformobilizationof89Kwasevalu- genicity island (PAI) designated 89K (Chen etal., 2007).
ated,anditsputativetransfermechanismwasinves- Recent genetic evidence showing that a two-component
tigated.Wereportthat89Kcanspontaneouslyexcise system (SalK/R) encoded within this island mediates a
to form an extrachromosomal circular product. The piglet-lethal phenotype has strengthened the case that
precise excision is mediated by an 89K-borne inte- 89KisaPAI(Lietal.,2008).
grase through site-specific recombination, with help As the name implies, 89K is approximately 89kb in
from an excisionase. The 89K excision intermediate length,flankedbyaperfect15bpdirectrepeat,andcon-
acts as a substrate for lateral transfer to non-89K tainsaputativephage-relatedintegrasegeneatits3′end.
These architectural features strongly suggest that it may
Accepted13January,2011.Forcorrespondence.*E-mailgaof@im. have been acquired by horizontal gene transfer (HGT).
ac.cn; Tel. [phone]; Fax [phone]; **E-mail
Meanwhile,theinternalmodularorganizationof89Kindi-
[email protected]; Tel. [phone]; Fax (+86) 23
68752834; ***E-mail [email protected]; Tel. [phone]; cates that it was generated by multiple recombination
Fax[phone]. events because it carries several unrelated gene blocks
Re-use of this article is permitted in accordance with the Terms
found in widely divergent species, making it difficult to
and Conditions set out at http://wileyonlinelibrary.com/onlineopen#
OnlineOpen_Terms ascertain its initial origin. A better understanding of the
©2011BlackwellPublishingLtd
T4SS-mediatedtransferof89KPAIinS.suis2 1671
molecularbiologyof89Kwillundoubtedlyprovideimpor- Table1. Conjugal transfer of 89K derivatives between S. suis 2
tant insight into the emergence, virulence, and evolution strains.
of this important pathogen. Indeed, it may reveal some
Transfer
clues concerning the molecular pathogenesis of STSS frequency
causedbyS.suis2. Donor Recipient (perdonor)*
It is well recognized that type IV secretion systems
05ZYH33-89KSpc SS2-NDrecA 5.4¥10-6
(T4SSs) play a key role in HGT, thus contributing to the 05ZYH33-89KSpc(DNaseI) SS2-NDrecA 4.9¥10-6
generation of genomic diversity, the evolution of new 05ZYH33-89KSpc(supernatant) SS2-NDrecA <10-8
05ZYH33-89KSpc S735::cat 6.0¥10-6
pathogenic strains, the dissemination of antibiotic resis-
05ZYH33-89KSpc 7996::cat 2.5¥10-6
tance genes and other virulence traits (Christie etal., 05ZYH33DmobA89K SS2-NDrecA <10-8
2005; Juhas etal., 2008). Based on the organization of CDmobA89K SS2-NDrecA 9.6¥10-5
05ZYH33DmobC89K SS2-NDrecA <10-8
genetic determinants, shared homologies and evolution-
CDmobC89K SS2-NDrecA 4.7¥10-6
aryrelationships,T4SSscanbeclassifiedintothreemajor 05ZYH33DvirB1-89K SS2-NDrecA 9.2¥10-7
types:(i)typeIVAsystemsresemblethearchetypalVirB/ CDvirB1-89K SS2-NDrecA 7.2¥10-6
05ZYH33DvirB4-89K SS2-NDrecA <10-8
VirD4systemofAgrobacteriumtumefaciensandarecon-
CDvirB4-89K SS2-NDrecA 3.8¥10-6
sideredtobetheparadigmofT4SS;(ii)typeIVBsystems 05ZYH33DvirB6-89K SS2-NDrecA <10-8
resemble the Dot/Icm system found in Legionella pneu- CDvirB6-89K SS2-NDrecA 4.3¥10-6
05ZYH33DvirD4-89K SS2-NDrecA <10-8
mophila and Coxiella burnetii; and (iii) otherT4SSs bear
CDvirD4-89K SS2-NDrecA 5.7¥10-6
littleornohomologytoTypeIVAorIVBsystems,andtheir 05ZYH33Dint SS2-NDrecA <10-8
functions await further investigation. Representative of CDint SS2-NDrecA 4.1¥10-6
05ZYH33Dxis SS2-NDrecA 8.7¥10-8
thethirdgrouparetherecentlydescribedgenomicisland
CDxis SS2-NDrecA 2.9¥10-6
(GI)-type T4SSs that are found in a broad spectrum of 05ZYH33Dhlc SS2-NDrecA <10-8
Gram-negativepathogens(Juhasetal.,2008).Ultimately, CDhlc SS2-NDrecA 6.5¥10-6
05ZYH33(pVA838-oriT) SS2-NDrecA 6.1¥10-4
it is the identification of the GI-type T4SSs that brings DmobA89K(pVA838-oriT) SS2-NDrecA <10-8
insight into the mechanisms by which GIs transfer
betweenbacteria(Juhasetal.,2009). *Thesevaluesarethemeansofthreeindependentexperiments.
Here,wepresentadetailedmolecularcharacterization
of the function and evolution of the 89K PAI in epidemic
isolates of S. suis 2. We provide genetic evidence transfertoanewhostspecies(Sakellarisetal.,2004;Qiu
showing that an 89K-borne integrase mediates site- etal.,2006;Coburnetal.,2007).Todeterminewhetherthe
specificexcisionandcircularizationof89K,withcontribu- 89KPAIistransmissiblefromadonorS.suisstraincon-
tionfromanauxiliaryproteinnamedexcisionase.The89K taining 89K to a recipient strain without this element, we
extrachromosomal circular excision product is self- performed conjugative mating experiments. To obtain a
transmissible and can conjugally transfer via a plasmid- marked version of 89K, a 3′ single cross-over mutant of
like process into S. suis recipient cells through the 89K- strain05ZYH33constructedinourpreviousworkwasused
encoded GI-typeT4SS transport conduit.Thus far, while as the donor, which was tagged with a spectinomycin
mobilizationofgeneticelementsbyT4SSshasbeenwell markerlocateddownstreamoftheSalK/Rtwo-component
studied in Gram-negative bacteria, the transfer of Gram- system within 89K (Li etal., 2008). We designated this
positive mobile genetic elements by T4SSs is currently derivative05ZYH33-89KSpcandthemodified89Kisland
limitedtoconjugativeplasmidsandtransposons.Without as 89K*. SS2-N, a virulent strain of S. suis 2 that does
a previous comprehensive study, we present the first not naturally contain 89K, was engineered to be
demonstrationofthehorizontaltransferofaPAImediated chloramphenicol-resistantbyreplacingtherecAgenewith
by a GI-type T4SS in a Gram-positive bacterium. We a cat gene cassette (creating SS2-NDrecA) and used as
suggest that this new mobile element is responsible, at the recipient. Medium containing both chloramphenicol
leastinpart,fortherecentSTSSoutbreaksinChinaand and spectinomycin was used to select exconjugants that
recent sporadic cases in neighbouring countries and received89K*.Inthefiltermatingassay,doubleantibiotic-
areas including Vietnam and Thailand (Holden etal., resistant transconjugants were obtained, and transfer of
2009),anditwillpossiblyspreadfurtherinthefuture. 89K*wasdetectedatameanfrequencyof5.4¥10-6per
donor (Table1). Using specific primers, the integrated
Results 89K* was detected in 10 randomly selected transconju-
gants,providingfurtherevidencethat89K*wastransferred
89KistransmissiblebetweenS.suis2strains
intotherecipients.Transferof89K*wasalsoobservedata
ItisnowwellestablishedthatPAIscomprisealargefamily similarfrequencybyusingotherS.suis2strainsasrecipi-
of mobile genetic elements and may undergo horizontal ents, including the virulent S735 strain and the avirulent
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
1672 M.Lietal. (cid:2)
7996strain,bothisolatedfromtheNetherlands(Table1). DuetotheaberrantGCcontentof89Kanditsabsence
However,conjugationexperimentsusingotherserotypes inthereferenceP1/7strainandanumberofinternational
of S. suis (serotypes 7 and 9) and other streptococcal virulentstrains,itappearsthat05ZYH33acquiredthe89K
species(S.sanguinisandS.agalactiae)asrecipientswere PAIhorizontally.89Kthenintegratedintothechromosome
performed but without success, suggesting that the 89K at the attB site in the rplL gene, generating two direct
transfermaybespecies-andserotype-specific. repeats(attLandattR)atbothendsoftheisland.Itiswell
documented that integrative and conjugative elements
(ICEs)areabletoexcisefromthechromosomestoform
Detectionofanextrachromosomalcircularformof89K
circular intermediates prior to conjugative transfer into
Toinvestigatetheunderlyingtransfermechanism,weper- recipientcells(Burrusetal.,2002).Weconfirmedthat89K
formedasystematicmoleculardissectionofthe89KPAI. did excise from the 05ZYH33 chromosome to produce a
Basedonourpreviouslyreportedcomparativesequence circular form and the host genome was repaired upon
analysis, 89K is located in the genome of S. suis strain excision because PCR products were obtained for both
05ZYH33 at a position between 05SSU0902 (hyd) and primer pairs P2/P3 and P1/P4 (Fig.2B). Subsequent
05SSU0983 (rplL), which encode a predicted hydrolase sequence analysis confirmed the expected structures of
andthe50SribosomalproteinL7/L12respectively(Chen attP and attB generated upon site-specific excision
etal., 2007). A total of 80 open reading frames (ORFs) (Fig.2C and D). Noticeably, the amplicons obtained with
wereannotatedinthe89Kisland,mostofthem(n=65)on primer pairs P2/P3 and P1/P4 showed a substantially
the minus strand. The overall gene organization in 89K weaker signal compared to that with P1/P2 and P3/P4,
is presented in Fig.1. Notably, 89K contains a Tn916 indicatingthat89Kexcisionisarareevent.Tomeasurethe
element, which is likely associated with the tetracycline rateof89Kexcision,wedevelopedareal-timequantitative
resistancephenotypebecausetheS.suis2referenceP1/7 PCR(qPCR)assayanddeterminedthatthefrequencyof
strainwithout89Kissensitivetotetracycline.Furthermore, 89K excision from an 05ZYH33 overnight culture was
89K carries a large lantibiotic biosynthesis cluster, which ~0.032%,aratesimilartothatobservedforSLGexcision
wassuggestedtobeincapableofproducingafunctional fromtheStreptomyceslividans66chromosome(Heetal.,
lantibiotic(duetomutation)butessentialforfullvirulence 2007).Thesedatademonstratedthat89Kcanspontane-
(PhelpsandNeely,2007;Holdenetal.,2009). ously excise from the chromosome and form a circular
Fig.1. Geneticmapofthe89KPAIintheS.suis2epidemicstrain05ZYH33.
A.AberrantaverageG+Ccontentof89K,whichismuchlessthanthatofthechromosome.
B.Geneorganizationof89K.TheterminaldirectrepeatsattLandattRareindicatedbyredtriangles.Genespredictedtolikelybeinvolvedin
themobilizationof89Kareshowninyellow.Formoregenedetails,seeChenetal.(2007).
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
T4SS-mediatedtransferof89KPAIinS.suis2 1673
Fig.2. Site-specificintegrationandexcisionof89KinthechromosomeofS.suis05ZYH33.
A.Schematicrepresentationofthesite-specificintegrationandexcisionof89K.Theleftandrightjunctions(attLandattR),whichareshown
asredrectangles,areformedbyrecombinationbetweenthechromosomalattBsiteandthe89KattPsite.Theflankinggenesareshownby
solidarrowsindicatingthedirectionoftranscription.Thelocationandorientationofprimersusedfordetectionofintegratedandexcised89K
areindicatedbythinarrows.
B.Detectionofacircularextrachromosomalformof89KandtheemptychromosomalattBsitefollowingpreciseexcision,byPCRanalysis
usingprimerpairswhicharepresenteduponthelanes,with05ZYH33genomicDNAasthetemplate.
C.PrintoutofthesequencingchromatogramofPCRproductsamplifiedwithprimerpairP2/P3,showingtheattPsite(boxed)formedbythe
joiningofthetwoendsof89K.
D.PrintoutofthesequencingchromatogramofPCRproductsamplifiedwithprimerpairP1/P4,showingtheemptychromosomalattBsite
(boxed)upon89Kexcision.
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
1674 M.Lietal. (cid:2)
excisionproduct.Detectionofbothintegratedandcircular
89K suggests that the populations of 05ZYH33 carrying
this island are heterogeneous. Most cells carry an inte-
gratedisland,whileotherscontainanextrachromosomal
circular one. Essentially, the same results were obtained
for98HAH12,anotherhighlypathogenicS.suis2strain,
which is predominantly responsible for the earlier 1998
STSSoutbreakinChina(datanotshown).
Theintgeneisessentialfor89Kexcision
A common feature of PAIs is the presence of a site-
specific recombinase that is presumably responsible for
the integration of the PAI into the bacterial chromosome
(Mantri and Williams, 2004). The 89K int gene, located
neartheleftendoftheisland(attL)atadistanceof63bp,
encodesa403-amino-acid(aa)integrasebelongingtothe
tyrosinerecombinasefamily,whichcatalysessite-specific
recombination between a chromosomal site (attB) and
a similar or identical sequence (attP) found on mobile
genetic elements including bacteriophages, insertion Fig.3. IntandXisarerequiredforefficient89Kexcision.
A.Geneticorganizationofthe3′terminalregionof89KinS.suis
sequencesandtransposons(EspositoandScocca,1997;
05ZYH33.Theintgeneandtheupstreamxisgeneareshownas
Nunes-Duby etal., 1998). Functional studies of related greyarrows(notdrawntoscale).Aputativeribosomebindingsite
members of this family give good reason to assume that (RBS)upstreamofxisisdepictedbyasmallflag.Thestem-loop
structurerepresentsaputativetranscriptionalterminator.The
89Kexcisionismediatedbythe89K-borneintegrase.To
positionofprimerP5isshown.
determine if this is the case, we generated an isogenic B.PCRamplicons(indicatedleft)obtainedfromthewild-type,
int knockout mutant of strain 05ZYH33 (creating mutantandcomplementedstrains(shownaboveeachlane).The
gyrAgeneservesasaninternalcontrol.
05ZYH33Dint)andexamined89Kexcisioninthismutant.
PCR products were generated using primer pairs P1/P5 faecalis (Fig.3A). The predicted xis is preceded by a
(primer P5 is located upstream of xis, as the int deletion putativeribosomebindingsite(AGGAG),whichislocated
removes the priming site for P2) and P3/P4. However, 8bp upstream from the initiation codon of xis. Further
negative results were obtained with primer pairs P3/P5 sequenceanalysisrevealedapalindromeof27bpwiththe
and P1/P4 (Fig.3B). qPCR analysis of the Dint mutant potential to form a stem-loop structure situated 85bp
alsodemonstratedthat89Kexcisionwaseliminated,with downstreamfromthestopcodonofint,whichmayfunction
attP not being detected. Trans-complementation of int as a transcriptional terminator. The genetic organization
restored the excision capacity of 89K to a level compa- of this locus suggests possible translational coupling
rabletothatoftheparentalstrain,withattPbeingpresent betweenxisandint.
in~0.025%ofcells.Theseresultssuggestthatdeletionof To ascertain whether xis is also involved in 89K exci-
intinstrain05ZYH33abolishedtheabilityof89Ktoexcise sion,theexcisionof89Kwasexaminedinanxisdeletion
fromthehostchromosome.Thus,Intisessentialfor89K mutant of strain 05ZYH33 (named 05ZYH33Dxis). As
excision and circularization by mediating site-specific shown in Fig.3B, PCR analysis of the excised circular
recombinationbetweentheattLandattRsites. 89Kandtherepairedchromosomeusingspecificprimers
both gave faint but still detectable signals in the Dxis
Identificationofanauxiliaryexcisionfactor,Xis mutant, suggesting that there is a very low level of Xis-
independent 89K excision. qPCR analysis revealed that
BasedonmostoftheICEsthathavebeenexamined,the
xisdeletiondecreasedthepresenceofattPto~0.0012%,
excisionfunctionofanintegraseisoftenstimulatedbythe
whilefunctionalcomplementationofxisrestoredtheexci-
presenceofacloselyadjacentrecombinationdirectionality
sion frequency to ~0.029%. These results demonstrated
factor(RDF),alsotermedanexcisionase(Xis),thatboth
that xis stimulates, but is not essential for, 89K excision
facilitates excision and inhibits integration (Sam etal.,
fromthechromosome.
2004).Inouranalysis,asmallORF(05SSU0904),located
immediatelyupstreamoftheintgeneandseparatedbya
RoleoftheGI-typeT4SSinlateraltransferof89K
14bp intergenic region, was found to encode a possible
Xishomologuewith32%aaidentitytoaputativeexcisio- In general, HGT occurs in bacteria through three main
nase(accessionnumberAAL27447.1)fromEnterococcus mechanisms: transformation, phage-mediated transduc-
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
T4SS-mediatedtransferof89KPAIinS.suis2 1675
tion and conjugation (Thomas and Nielsen, 2005). The DvirB6-89K and DvirD4-89K were used as donors.
likelihood of transformation was excluded based on our However,aDvirB1-89Kderivativeof89Kwasstillableto
observationthatuseofDNaseIinthefiltermatingassay transferatafrequencyof9.2¥10-7,approximatelysixfold
did not abolish the transfer ability of 89K (Table1). Fur- less than that of 89K*.Asimilar phenomenon had been
thermore, no 89K transfer was detected when the donor reported that the transfer capacity of the IncQ plasmid
culture supernatant was incubated with the recipient, RSF1010 was reduced approximately 10-fold in a virB1
which also ruled out the possibility of phage-mediated mutant(Bohneetal.,1998).Allofthesetransferdeficien-
transduction(Table1).Therefore,conjugationisthemost cies could be fully or partially restored by trans-
likelymechanismforinter-straintransferof89K. complementation (Table1).Thus, these results indicated
Extensive sequence analysis of 89K revealed several that the GI-type T4SS genes are absolutely required for
ORFs whose putative products show similarity to conju- 89Ktransfer,withtheexceptionofvirB1-89K(whichcon-
gation and mobilization proteins involved in conjugative tributestobutisnotessentialfor89Ktransfer).
DNAtransfer(Fig.1B).Onthe3′halfofthe89Kisland,a Parallelconjugativematingexperimentswerealsoper-
peptide encoded by 05SSU0913 showed similarities to formedtoevaluateifthegenesproposedtobeinvolvedin
various prokaryotic DNA relaxases that initiate conjugal theexcisionandcircularizationof89Karealsoinvolvedin
transferbystrand-andsite-specificcleavageatthetrans- thetransferprocess.AsshowninTable1,anintdeletion
ferorigin(oriT),suchastheVirD2component(accession mutantwastransfer-deficientasexpected,whilethetrans-
numberNP_059814.1)oftheA.tumefaciensT4SS(36% fer frequency of the Dxis 89K derivative was reduced by
aa identity) and the MobA relaxase (accession number almosttwoordersofmagnitude,proximaltothelowerlimit
AAA26445.1) of the broad host-range plasmid R1162 of detection in our mating system. These observations
(39%aaidentity).Peptide(05SSU0914),locatedadjacent suggestthatthe89K-encodedintegraseandexcisionase
tothisrelaxase,shared36%aaidentitytotheaccessory likelycooperativelygeneratetheextrachromosomalcircu-
proteinMobC(accessionnumberAAA26444.1)ofR1162, larformof89K,whichrepresentsanintermediaterequired
whichisproposedtoactasamolecularwedge,denatur- forthetransferprocessitself.
ing oriT strands and providing a single-stranded (ss)
substrate for the relaxase (Zhang and Meyer, 1997).
89KhasafunctionaloriTsite
Therefore, 05SSU0913 and 05SSU0914 were accord-
inglydesignatedmobA89KandmobC89Krespectively. Thetransferofplasmidsbetweenbacterialcellsbyconju-
Ontheotherhalfof89K,fourgeneproductsdisplayaa gationistheresultoftwoprocesses:(i)mating-pairforma-
similarities to the Agrobacterium T4SS components. tion between the donor and recipient, and (ii) a DNA
05SSU0968 is a VirB1-type lytic transglycosylase (18% processingreactionthatpreparestheplasmidfortransfer
identitytotheT4SSVirB1component,accessionnumber (WillettsandWilkins,1984;PansegrauandLanka,1996).
NP_059799.1), 05SSU0969 is a putative VirB4-like Conjugation can proceed only after the formation of a
ATPase (41% identity to the T4SS VirB4 component, protein-DNAcomplex(relaxosome)attheoriTsite,which
accession number NP_059802.1), 05SSU0971 is a is often located in the intergenic region upstream of its
hypotheticalVirB6-likechannelsubunit(12%identitytothe cognaterelaxasegene(Franciaetal.,2004).TheoriTof
T4SS VirB6 component, accession number NP_ the 89K PAI was predicted to be a sequence of ~40nt
059804.1), and 05SSU0973 is a putative VirD4-like cou- positionedwithinthe05SSU0915codingsequence,which
plingprotein(40%identitytotheT4SSVirD4component, ispredictedtoencodeapolypeptideofunknownfunction
accession number NP_059816.1). Thus, 05SSU0968, with62%aaidentitytoahypotheticalprotein(SMSK597-
05SSU0969, 05SSU0971 and 05SSU0973 were accord- 1197, accession number ZP_07642092.1) of S. mitis
ingly designated virB1-89K, virB4-89K, virB6-89K and SK597. This segment contains a perfect 9nt inverted
virD4-89Krespectively.Basedonthesepredictions,itwas repeatsequence(predictedbytheDNAStriderprogram)
logical to assume that horizontal transfer of the 89K PAI that may be necessary to form a stable nucleoprotein
mayoccurthroughaGI-typeT4SS-mediatedconjugation complex during conjugation (Bhattacharjee and Meyer,
process. 1993). The precise nic (nickase cleavage) site was
Todetermineifthe89KPAIharboursafunctionalGI-type predicted to reside at position 3′-(887140)CTCG/
T4SS, mobilization experiments were performed to CAAA(887147) on the negative strand, which is located
examineifthetransferabilityof89Kdecreasedbymaking 8nt downstream of the inverted repeats within the oriT
individual knockouts of the T4SS gene homologues. region based on an extensive sequence alignment with
Table1 shows the results of conjugation experiments otheroriTsitesthathavebeenexperimentallydetermined
between these T4SS gene homologue mutants and the previously.Figure4showstheoverallgeneorganizationof
recipient strain SS2-NDrecA. No transconjugants were the DNA processing region of 89K. We noticed that
obtained when DmobA89K, DmobC89K, DvirB4-89K, MobA89KhasanN-terminalrelaxasedomainbutlacksa
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
1676 M.Lietal. (cid:2)
Fig.4. FunctionalorganizationoftheDNA-processingregionof89K.Themobilizationgenes(mobA89KandmobC89K)involvedintheDNA
processingaremarkedbyopenarrows(notdrawntoscale).Theshadedregionsindicatetheconserveddomainsencodedtherein,withthe
internalconsensusmotifsindicatedbyblackrectangles.Theasteriskrepresentstheconservedmotif(L/FxxxG/SxNxNQxAxxxN)withinthe
MobCfamily.The89KoriTwaspredictedtobepositionedwithinthe05SSU0915ORF,whichencodesahypotheticalproteinofunknown
function.Thedirectionoftransferisshownbyahorizontalarrow.Thelowerportionofthisfigureshowsthealignmentofthe89KoriTsitewith
otheroriTregionsofseveralmobilizableplasmids(indicatedleft).Arrowsabovethesequencesrepresentthelocationsofinvertedrepeats,
andverticalarrowsshowexperimentallydeterminednicksites.
C-terminal helicase domain, which usually possesses a greater than that of 89K*. PCR analysis confirmed the
processive 5′ to 3′ unwinding activity that provides the efficient mobilization of pVA838-oriT by using specific
motiveforceforstrandtransfer(Llosaetal.,1996;Matson primers. However, no transconjugants were obtained
etal., 2001). Instead, we found that ORF 05SSU0934 when the DmobA89K mutant was used as the carrier
within89KencodesamatchtotheDNAhelicase(acces- donor. These results demonstrated that the non-
sion number ACF56805.1) of S. pneumoniae G54, with conjugative plasmid pVA838 could be mobilized only by
93%aaidentity.Invivomutationalanalysisdemonstrated 05ZYH33 carrying an active relaxase after introducing a
thatthisgene(designatedhlc,helicase)isnotrequiredfor fragment encompassing the putative 89K oriT. In other
89Kexcisionandcircularization(Fig.3B)butisessential words, the 89K PAI has a functional oriT site recognized
for89Ktransfer(Table1). byitscognaterelaxase,MobA89K.
To test the hypothesis that the putative 89K oriT
sequence plays a crucial role in the initiation of conjugal
MobA89KanditsN-terminalrelaxasedomaindisplay
transfer, a 355bp segment containing the putative oriT
specificnickingactivityinthepresenceoftheMobC89K
site was cloned into a non-transmissible Escherichia
accessoryproteininvitro
coli–S. suis shuttle plasmid, pVA838. The recombinant
plasmid(pVA838-oriT)wasfoundtobeabletotransferat To gain insight into the relaxation process of 89K,
a frequency of about 6.1¥10-4 transconjugants per His -tagged MobA89K, MobC89K and the first 258 aa of
6
donor, which is approximately two orders of magnitude MobA89K (designated MobA89KN258) were expressed
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
T4SS-mediatedtransferof89KPAIinS.suis2 1677
Fig.5. Site-andstrand-specificrelaxationofthe89KoriTregion.
A.PhysicalmapofthepMD-oriTplasmid,whichcontainstheoriTregionof89KandservesassubstrateDNAintherelaxationanalysis.The
putativeoriTnicksite(nic)isindicatedbyaverticalarrow.
B.EquivalentsubstratepMD-oriTplasmidwasincubatedwith(+)orwithout(–)theproteinsofinterest.Reactionproductswereanalysedby
standardagarosegelelectrophoresis(0.9%).OC,opencircularplasmidDNA;CCC,covalentlyclosedcircularplasmidDNA.
C.Schematicrepresentationoftheexpectedsingle-strandspeciesgeneratedbyasite-specificnick(arrow)onendonuclease-linearized
pMD-oriTDNA.ThesizeofeachssDNAfragmentisshown.
D.pMD-oriTwaslinearizedwitheitherScaIorNdeI,andtherelaxationmixtureswereanalysedona0.9%alkalineagarosegel.Lanes:1,
ScaI-linearizedpMD-oriTwithMobA89KandMobC89K;2,NdeI-linearizedpMD-oriTwithMobA89KandMobC89K;3,ScaI-linearized
pMD-oriTwithMobA89KN258andMobC89K;4,NdeI-linearizedpMD-oriTwithMobA89KandMobC89K.The1kbDNAladdermarkeris
showntotheright(M).
and purified. When overexpressed at low temperature pMD-oriTwasusedassubstrateDNAinaninvitrocleav-
(16°C), MobA89K and MobC89K were both mainly age reaction. As shown in Fig.5B, no relaxation was
presentinthesolublefractions.Althoughamajorityofthe observed in the absence of either protein component.
MobA89KN258proteinwaspresentintheinclusionbody However,MobA89Kshowedanobviousrelaxationactivity
fractions, sufficient soluble material was produced to inthepresenceoftheaccessoryproteinMobC89K,yield-
recover useful amounts of active protein. The molecular ing 56% nicked, open circular (OC) plasmid DNA com-
masses of the expressed recombinant proteins agreed pared with 10% in the untreated control. These findings
well with those predicted. All proteins were purified to suggestedthatbothMobA89KandMobC89Karerequired
>95%homogeneityaftermetalionchelatingchromatog- for the nicking reaction. Meanwhile, MobA89KN258 also
raphyandgelfiltration(Fig.S1). increasedtheconversionofthecovalentlyclosedcircular
Relaxosomesarepresumablycomposedofsupercoiled (CCC) plasmid DNA into the relaxed form (OC) by the
plasmid DNAwith transfer factors complexed at the oriT addition of MobC89K, yielding 45% nicked products (i.e.
site (Lanka and Wilkins, 1995). To confirm whether the slightlylessthanthatobservedforthefull-lengthMobA89K
MobA89KandMobC89Kproteinscaninducethesite-and protein).
strand-specific DNA cleavage reaction, the relaxation In another set of experiments, linearized plasmid DNA
activitiesofthesepurifiedproteinsonsupercoiledplasmid was subjected to nic site analysis using denaturing
DNAweretested.ThesupercoiledoriT-containingplasmid agarosegels.TheoriT-containingplasmidpMD-oriTwas
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
1678 M.Lietal. (cid:2)
linearized with a restriction endonuclease (ScaI or NdeI) Chromosomal transfer mediated by this kind of Hfr-like
and then co-incubated with the protein components of mechanism was regarded as the major evolutionary
interest.ThepredictedoriTnickoneachofthesespecifi- force driving the genome dynamics of S. agalactiae
cally linearized fragments is shown in Fig.5C. As (Brochet etal., 2008).
expected, three distinct bands corresponding to the pre- A combination of bioinformatics and functional analy-
dicted sizes (based on nicking at the predicted oriT site) ses revealed a novel member of the GI-type T4SS
were detected on an alkaline agarose gel, in which the family that plays a key role in the conjugal transfer of
dsDNAsweredenaturedintosinglestrands(Fig.5D).The 89K. Throughout the past half century, investigations of
C-terminally truncated MobA89K protein showed a com- T4SSs have largely focused on defining the mecha-
parablenickingactivitytothatofthefull-lengthprotein.In nisms of action of several model systems of Gram-
all cases, the largest fragment corresponded to the full- negative bacteria, such as the F (IncF), R388 (IncW),
length plasmid ssDNA, and the different sizes of the two RP4 (IncP) and pKM101 (IncN) plasmid conjugation
additional bands with higher mobility indicated site- systems and the A. tumefaciens VirB/VirD4 system.
specific cleavage of one of the plasmid strands. Indeed, However, little is known about the architectures or
the sum of the sizes of these two fragments equals one mechanisms of T4SSs in Gram-positives (Alvarez-
full-length linear ssDNAplasmid. These results, together Martinez and Christie, 2009). Although T4SS-mediated
with the supercoiled plasmid DNA cleavage data mobilization of genetic elements has been characterized
described above, demonstrated that the in vitro nicking in some species of Gram-positive bacteria, such ele-
reaction was dependent on the presence of both ments are currently limited to conjugative plasmids (e.g.
MobA89K and MobC89K, and the MobA89K-dependent E. faecalis pCF10, S. agalactiae pIP501 and C. perfrin-
oriT nicking activity resides within the N-terminal 258-aa genspCW3)andtransposons(e.g.E.faecalisTn916,B.
relaxasedomainofMobA89K. subtilis ICEBs1 and S. agalactiae TnGBS2). Indeed,
transferofGram-positiveGIs(especiallyPAIs)byT4SSs
has not yet been reported. Our findings expand the rep-
Discussion
ertoire of Gram-positive ICEs that can be mobilized by
Integrase-mediated precise excision of mobile elements T4SS, and this is the first characterization of horizontal
from the chromosome typically represents the first step transfer of a Gram-positive PAI by a GI-type T4SS. In
in the lateral transfer process. Such excision events contrast, GI-type T4SSs have been identified in various
usually lead to the formation of circular episomal prod- Gram-negative GIs such as pKLC102, PAPI-1, SPI-7
ucts that are intermediates in the packaging of some and the Pseudomonas clc element, as well as others
ICEs (e.g. P4) into phage particles (Bowden and derived from a wide variety of Gram-negative plant and
Modrich, 1985), or serve as substrates for conjugative animal pathogens, including Erwinia carotovora (Chen
transfer of other elements, such as PAPI-1 (Qiu etal., etal., 2008), Xylella fastidiosa (Zaini etal., 2008), Pho-
2006) and SXT (Burrus and Waldor, 2003). Sequence torhabdus luminescens (Held etal., 2007) and Yersinia
analysis revealed that 89K is not similar to an integrated pseudotuberculosis (Collyn etal., 2006). The complete
prophage, suggesting that the excised circular 89K may set of 24 genes thought to encode the putative GI-type
undergo lateral transfer. Confirmation of this hypothesis T4SS in H. influenzae ICEHin1056 has been fully or
was obtained by the observation that transfer of a largely identified in the GIs of the above-mentioned
SpcR-marked 89K occurred at a frequency of approxi- Gram-negative pathogens, suggesting that GI-type
mately 10-6 per donor, similar to that of the Pseudomo- T4SSs are highly conserved among these species
nas clc element (Sentchilo etal., 2003) and the (Juhas etal., 2008). Comparative genomic and func-
Salmonella SGI1 island (Doublet etal., 2005). Further, tional analysis suggested that 89K encodes a GI-type
89K transfer absolutely requires the 89K-borne inte- T4SS at its 5′ terminus (05SSU0961-05SSU0982), dif-
grase, while a Dxis derivative can still transfer at a low fering in gene content and organization with the Gram-
but measurable frequency. This suggests that 89K exci- negative counterparts (Fig.S2). This indicates that there
sion is required for its transfer, and Xis may exert its may be an evolutionary divergence of GI-type T4SSs
effect by reducing the number of copies of excised between Gram-negative and Gram-positive bacteria,
circularized 89K molecules. Notably, Gilmore and perhaps due to the differences in cell wall structure.
co-workers demonstrated that transfer of the enterococ- Based on our experimental results presented here, in
cal PAI did not require the PAI-resident integrase, exci- combination with previous studies on the Agrobacterium
sionase or putative conjugation genes. Instead, transfer oncogenic T-DNA transfer system (Christie and Vogel,
only occurred from donors possessing a pheromone 2000; Atmakuri etal., 2004; Middleton etal., 2005; Guo
responsive-type of conjugative plasmid via an Hfr-like etal., 2007), we would like to propose a model for the
and RecA-dependent mechanism (Manson etal., 2010). transfer mechanism of the 89K PAI (Fig.6). In a small
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683
T4SS-mediatedtransferof89KPAIinS.suis2 1679
Fig.6. Hypotheticalmodelforthefunctionofthe89KGI-typeT4SS.
The89K-borneintegrasemediatespreciseexcisionof89Kfromthechromosomewiththeaidofexcisionase.Afterreceivingamatingsignal,
thetransferinitiateswiththeoriTnickingreactioncatalysedbytheMobA89KandMobC89Kproteins.Thecleavedstrandisthenunwoundby
thehelicaseanddeliveredtothe89K-encodedGI-typeT4SSmachinery.TheVirB1-89Kcomponentisthoughttoactasalytic
transglycosylase,locallyopeningthepeptidoglycanwithcontributionsfromsomeotherfactor(s)duringtheassemblyofthetransportchannel.
VirD4-89KisacouplingproteinrequiredtodelivertheDNAsubstratetothetransportchannel,andtogetherwithVirB4-89K,requiredto
energizethesubstratetransportacrossthechannel.ThehighlyhydrophobicVirB6-89Khomologueconstitutesthemainportionofthe
transportchannel.
proportionofhostbacteria,the89K-borneintegrasemedi- helicase unwinds the nicked strand during relaxosome
atespreciseexcisionof89Kfromthechromosomewiththe formation, and the VirD4-89K coupling protein then links
helpofanexcisionase.Upondetectionofamatingsignal, therelaxosomeandleadsitdirectlytothematingchannel
the transfer of the excised circular intermediate of 89K (Llosaetal.,2003).TheVirB1-89Klytictransglycosylase,
begins (Llosa etal., 2002). In the first step, MobA89K forwhichpeptidoglycancleavageactivityhasbeenprevi-
initiates the processing reaction by generating a strand- ouslydemonstratedinA.tumefaciens(Zupanetal.,2007),
andsite-specificnickattheoriTsiteof89K,withhelpfrom shouldplayacrucialroleinlocalopeningofthepeptidogly-
theauxiliaryMobC89Kprotein.Next,the05SSU0934DNA canduringtheassemblyofthetransenvelopetransporter
©2011BlackwellPublishingLtd,MolecularMicrobiology,79,1670–1683