Evolution of Conjugation and Type IV Secretion Systems Article

17 Pages·2013·0.59 MB·English

Evolution of Conjugation and Type IV Secretion Systems
Julien Guglielmini,*,1,2 Fernando de la Cruz,3 and Eduardo P.C. Rocha1,2
1De´partementGe´nomesetGe´ne´tique,MicrobialEvolutionaryGenomics,InstitutPasteur,Paris,France
2CNRS,UMR3525,Paris,France
3DepartamentodeBiolog´ıaMoleculareInstitutodeBiomedicinayBiotecnolog´ıadeCantabria(IBBTEC),Universidadde
Cantabria-CSIC-SODERCAN,Santander,Spain
*Correspondingauthor:E-mail:[email protected].
Associateeditor:HowardOchman
Abstract
Geneticexchangebyconjugationisresponsibleforthespreadofresistance,virulence,andsocialtraitsamongprokaryotes.Recent D
o
worksunraveledthefunctioningoftheunderlyingtypeIVsecretionsystems(T4SS)anditsdistributionandrecruitmentforother w
n
biological processes (exaptation), notably pathogenesis. We analyzed the phylogeny of key conjugation proteins to infer the lo
a
d
evolutionaryhistoryofconjugationandT4SS.Weshowthatsingle-strandedDNA(ssDNA)anddouble-strandedDNA(dsDNA) e
d
conjugation, while both based on a key AAA+ ATPase, diverged before the last common ancestor of bacteria. The two key fro
ATPasesofssDNAconjugationaremonophyletic,havingdivergedatanearlystagefromdsDNAtranslocases.Ourdatasuggest m
h
thatssDNAconjugationarosefirstindidermbacteria,possiblyProteobacteria,andthenspreadtootherbacterialphyla,including ttp
s
bacterialmonodermsandArchaea.IdentifiableT4SSfallwithintheeightmonophyleticgroups,determinedbybothtaxonomy ://a
andstructureofthecellenvelope.Transfertomonodermsmighthaveoccurredonlyonce,butfolloweddiverseadaptivepaths. ca
d
Remarkably,someFirmicutesdevelopedanewconjugationsystembasedonanatypicalrelaxaseandanATPasederivedfroma e
m
dsDNA translocase. The observed evolutionary rates and patterns of presence/absence of specific T4SS proteins show that ic
.o
conjugation systems are often and independently exapted for other functions. This work brings a natural basis for the classi- u
p
ficationofallkindsofconjugativesystems,thustacklingaproblemthatisgrowingasfastasgenomicdatabases.Ouranalysis .co
m
providesthefirstglobalpictureoftheevolutionofconjugationandshowshowaself-transferrablecomplexmultiproteinsystem /m
hasadaptedtodifferenttaxaandoftenbeenrecruitedbythehost.Asconjugationsystemsbecamespecifictocertaincladesand b
e
cellenvelopes,theymayhavebiasedtherateanddirectionofgenetransferbyconjugationwithinprokaryotes. /artic
Keywords:bacterialconjugation,horizontalgenetransfer,typeIVproteinsecretion,exaptation,plasmidevolution. le
-a
b
s
tra
DNA(ssDNA)versusdouble-strandedDNA(dsDNA),andin c
Introduction thecomplexityofthetransportsystem(delaCruzetal.2010; t/30
/2
Prokaryotic genomes adapt quickly to new environmental Vogelmannetal.2011).Bothtypesofconjugativesystemsare /3
A1
conditions largely because they can acquire pre-evolved eitherencodedbyautonomouslyreplicatingplasmidsorin- 5
traits by horizontal gene transfer (HGT) (de la Cruz and serted in chromosomes as integrative conjugative elements r/10
t1
3
Davies 2000; Gogarten et al. 2002; Ochman et al. 2005). (ICEs) (Smillie et al. 2010; Wozniak and Waldor 2010). We i9
c8
Conjugation is a mechanism of genetic transfer that allows recentlymadealarge-scaleidentificationofssDNAconjuga- le1 by
single-event transfer of large DNA fragments, up to entire tionsystems,bothinplasmidsandICEs,andfoundthemto g
u
chromosomes. Conjugation can transfer nonhomologous beessentiallyshort-termvariantsofotherwiseidenticalback- e
s
genes to the recipient genome and has a broader host boneelements(Guglielminietal.2011). t o
n
range than transduction or transformation (Amabile- In the following, we note proteins from a given genetic 1
1
Cuevas and Chicurel 1992; Llosa et al. 2002; Chen et al. elementbyGI ,whereGIreferstothegeneidentification A
MGE p
2005).Accordingly,recentworksuggeststhatconjugationis andmobilegeneticelement(MGE)tothenameoftheelem- ril 2
the most frequent mechanism of HGT (Halary et al. 2010). ent (e.g., TraCF corresponds to the TraC protein of the F 019
Indeed,conjugativesystemsaremajorplayersinthespreadof plasmid).ConjugativesystemsinvolvedinssDNAconjugation
antibiotic resistance, metabolic pathways, symbiotic traits, includetwomajorproteincomplexes:relaxosomesandtype
and other mobile genetic elements (de la Cruz and Davies IVsecretionsystems(T4SS)(reviewedinFronzesetal.2009;
2000;Thomas2000;vanderMeerandSentchilo2003;Frost delaCruzetal.2010).MGEdeliverythroughthemembranes
et al. 2005; Ding and Hynes 2009; Allen et al. 2010). ofthedonorandrecipientcellsisdonebytheT4SS(fig.1).In
Conjugation is also involved in the establishment of social Proteobacteria,theT4SSarealargeproteincomplex,includ-
processes, promoting biofilm formation (Ghigo 2001) and ing a ubiquitous ATPase (VirB4 or the distant homolog
Ti
spreadingofcooperativetraits(Nogueiraetal.2009;Rankin TraU ), mating-pair formation (MPF) proteins that
R64
etal.2011).Therearetwoknownmodesofconjugationthat form the transport channel, and a pilus that attaches to
differbothinthetypeoftranslocatedDNA,single-stranded the recipient cell (Alvarez-Martinez and Christie 2009;
(cid:2)TheAuthor2012.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://
creativecommons.org/licenses/by-nc/3.0/),whichpermitsnon-commercialreuse,distribution,andreproductioninanymedium, Open Access
providedtheoriginalworkisproperlycited.
Mol.Biol.Evol.30(2):315–331 doi:10.1093/molbev/mss221 AdvanceAccesspublicationSeptember13,2012 315
MBE
Guglielminietal. . doi:10.1093/molbev/mss221
usingATPtotranslocatethecomplexacrosstheinnermem-
brane(Gomis-Ruthetal.2004;Tatoetal.2005).Themajority
of T4CPsbelong to the VirD4 family, but some T4SS were
B2/B5 Ti
recently found to lack VirD4 and instead use a distantly
related ATPase as T4CP (TcpA ) (Parsons et al. 2007;
pCW3
OM B7/B9
Steen et al. 2009). Protein secretion systems based on T4SS
donotrequirerelaxosomes.TheyusuallyrequireT4CP,albeit
exceptions have been found in Bordetella pertussis and
B1 B8 B10
Brucella spp. (Alvarez-Martinez and Christie 2009). In these
systems,proteinsaretranslocatedacrosstheinnermembrane
B6 byothermeans.
IM Conjugation of dsDNA takes place in mycelia-producing
B3/B4 Actinobacteria(Grohmannetal.2003;Ghinetetal.2011).It D
o
B11 relies on a single protein: TraB that translocates dsDNA w
pSG5 n
betweenneighboringcellsinmycelia(Possozetal.2001).This lo
D4 a
d
protein resembles, in sequence and function, the essential e
MOB protein FtsK that segregates sister chromosomes in the last d fro
m
stages of chromosomal replication (Bigot et al. 2007;
FIG. 1. Scheme of the most-studied T4SS, the vir system of h
A.tumefaciensTiplasmid.TheVirBXproteinsaredepictedasBX(e.g., Vogelmann et al. 2011). They are both members of the ttp
s
B5referstotheVirB5protein).ThecouplingproteinVirD4(D4)andthe AAA+ motorATPasefamily,whichalsoincludesbothtypes ://a
mobilizationcomplex,whichincludestherelaxase(MOB)-DNAcom- ofT4CP(VirD4andTcpA)andbothtypesofATPasesessen- ca
d
plexarealsorepresented.OM:outermembrane;IM:innermembrane. tialforthefunctionofT4SS(VirB4andTraU).Hence,allkey em
proteins of the dsDNA and ssDNA conjugation systems are ic.o
Fronzes et al. 2009). The large (>70kDa) VirB4 ATPase is evolutionarilyrelated.Thisassociationhasnotyetbeenclar- up
.c
highly conserved in sequence and the only protein with ifiedfromaphylogeneticpointofview. o
m
clear-sequence homologs in all known T4SS. It is therefore T4SSareoftenrecruitedbybacterialpathogenstodeliveref- /m
b
themarkerofthepresenceofaT4SS(Alvarez-Martinezand fectorstoeukaryoticcells(Weissetal.1993;Vogeletal.1998; e
/a
Christie 2009). VirB4 is thought to energize the assembly or Seubertetal.2003;Nystedtetal.2008).TheseMOBlessT4SS, rtic
activity of the secretion channel and is essential for pilus called so because they do not contain a relaxase gene, are le
-a
biogenesis and substrate transfer (Berger and Christie 1993; closely related to the T4SS of conjugative systems. Indeed, b
s
Fullner et al. 1996; Wallden et al. 2012). Four MPF families severalT4SScanperformbothconjugationbetweenbacteria tra
c
have been described in Proteobacteria: MPF (based on the and protein delivery (Vogel et al. 1998; Llosa et al. 2003; t/3
T 0
T-DNA conjugation system of A. tumefaciens plasmid Ti), Schroderetal.2011).ProteindeliverybyT4SSisessentialfor /2
/3
MPF (basedonplasmidF),MPF (basedontheIncIplasmid thevirulenceofmanyplantandanimalpathogens,including 1
F I 5
R64),andMPFG(basedonICEHIN1056)(Smillieetal.2010). Legionella pneumophila, Helicobacter pylori, Bartonella spp., /101
Thesefourmodelsdescribeallfunctionallystudiedandnearly Coxiella burnetii, and A. tumefaciens (reviewed in Seubert 3
9
8
all T4SS identified by bioinformatic methods among et al. 2003; Juhas et al. 2008; Alvarez-Martinez and Christie 1
b
Proteobacteria, both in plasmids and chromosomes 2009).OnlyT4SSamongMPFTandMPFIhavebeenexperi- y g
(Guglielmini et al. 2011). The best-studied system is the vir mentallyshowntobeusedforproteindelivery.Theextreme ue
s
operon (MPFT) from A. tumefaciens Ti plasmid. This small flexibilityofT4SShasallowedatleasttwoothertypesofex- t on
operon encodes 11 VirB proteins (Thompson et al. 1988; aptations, i.e., evolutionary events in which part of the 1
1
Ward et al. 1988), and we use these names as a template pre-existing machinery of conjugation was recruited for A
p
for naming the protein families of the MPFT system. T4SS other functions (Gould and Vrba 1982). H. pylori genomes ril 2
from Cyanobacteria, Bacteroides, Firmicutes, Actinobacteria, encode a MOBless T4SS that is used for natural transform- 0
1
9
andArchaeahavehomologstoVirB4(Guglielminietal.2011). ation.ItisnecessarytoimportenvironmentalDNA(Hofreuter
ssDNA-conjugative systems are very diverse, but very few etal.2001).InNeisseriagonorrhoeae,oneT4SSisresponsible
studieshavebeendoneonthestructure,function,andevo- for DNA export to the extracellular space, an intermediate
lutionofT4SSoutsideProteobacteriaandFirmicutes. step in the process of natural transformation among these
ThetwootheressentialcomponentsofthessDNAconju- bacteria (Hamilton et al. 2005). Interestingly, in the case of
gation machinery are the relaxosome and the type IV cou- Neisseria,thelocusencodesaT4SSandaMOB -typerelaxase
H
pling protein (T4CP). The relaxosome is composed of the thatisnecessaryforDNAexport(Salgado-Pabonetal.2007).
relaxase (MOB) and often includes auxiliary proteins. It ApreviousanalysisofMPF systemssuggeststhatexaptation
T
nicks the dsDNA and binds the resulting ssDNA at the of conjugative systems occurred several times in evolution
originoftransfer.Thediversityandevolutionofthedifferent (Franketal.2005).BecausewerecentlyfoundthatMOBless
families of relaxases has been extensively studied (Garcillan- T4SSaresignificantlymoreabundantthanpreviouslythought
Barcia et al. 2009). The highly conserved T4CP binds the (Guglielminietal.2011),thispointneedstobereassessedfor
DNA-relaxase substrate and couples it to the T4SS, possibly MPF anddevelopedforotherMPFtypes.
T
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EvolutionofConjugationandT4SS . doi:10.1093/molbev/mss221
Although studies on conjugation are as old as molecular presence/absenceofthedifferentcomponentsofthevirsys-
biology itself (Lederberg and Tatum 1946), several recent tem, we made additional protein profiles, namely for VirB1,
works have significantly changed our understanding of this VirB2,VirB5,VirB7,VirB10,andVirB11.WefirstusedPSI-Basic
process.Theseincludethediscoveryofnewconjugationsys- Local Alignment Search Tool (BLAST) (e value<0.1) to
tems(Juhas,Crook,etal.2007),ofnewkeyelementsinknown search for distant homologs, using as query each of these
conjugation systems, e.g., TcpA (Parsons et al. 2007) and of genesfromtheVirBlocusoftheA.tumefaciensplasmidpTi
theimportantrole of ICEs(Burrus etal. 2002;Wozniak and SAKURA(RefseqentryNC_002147)andtheaforementioned
Waldor 2010). Recent functional studiesexploredthe diver- databankofcompletelysequencedreplicons.Giventheprob-
sity of T4SS (Alvarez-Martinez and Christie 2009), and bio- lems of convergence of PSI-BLAST when using complete
informatics work unraveled the presence of T4SS in several genomes,andtheextensivesimilarityofplasmidandchromo-
newclades(Guglielminietal.2011).Finally,otherworkshigh- somal conjugative systems (Guglielmini et al. 2011), we re-
lighted the close structural and functional relationship be- stricted homology searches to plasmid sequences when
tween T4SS used for protein secretion and conjugation building protein profiles. We retrieved the proteins with D
(Fernandez-Gonzalez et al. 2011). This succession of works hits for each protein family and built multiple alignments ow
n
openstheopportunitytoinferaglobalscenariofortheevo- using MUSCLE (Edgar 2004). We removed the few proteins lo
a
lutionofconjugativesystemsandT4SS,whichisthegoalof withsizesverydifferentfromtheaverage.Wethenrebuiltthe de
d
thepresentwork.Toassesstheuncertaintyinthephylogen- multiple alignments with MUSCLE and trimmed them to fro
eticreconstruction,weusedclassicalmethodssuchasboot- remove the sites at the edges that were poorly aligned. We m
h
strapanalyses.Yet,becausetheselargeanddeepphylogenetic used HMMER 3.0 (Eddy 2011) to produce hidden Markov ttp
reconstructionscanbesensitivetoalignmentalgorithmsand model(HMM)profilesandtoperformsearcheswithingen- s://a
to methods to extract informative positions (Philippe et al. omes.IntheanalysisoftheevolutionoftheMPF system,we c
T a
2011),wealsotestedtherobustnessofourresultsbycompar- onlyconsideredthehitsthatcolocalizedwithpreviouslyde- de
m
ingthemwithtwoautomaticanalysesthatwedidinparallel. tected vir proteins (VirB3, VirB4, VirB6, VirB8, VirB9). FtsK ic
.o
Toguidethecomparisonsbetweenthethreesetsofanalyses, proteinswereretrieveddirectlybyusingthePFAMPF01580 u
p
wemadeanassessmentofthequalityofthemultiplealign- profile. TraB proteins, being closely related to FtsK, were .co
m
mentsusingT-Coffee(Notredameetal.2000).Bydefault,we retrievedbyBLASTPsearchesofTraBfromStreptomycesplas- /m
onlymentiontheresultsofourexpertanalysis(typically,the midpCQ3(YP_003280879)ontheActinomycetalesproteins b
e
obentewweeitnhmhiegthheostdsalwignhemnetnhteqyuaarleityr)e,lebvuatnht.igThhlieghotvderifaflelrsetnrucecs- ftrhoemnbtuhieltRaepfsreoqteidnaptarboafislee.foWrethsisamprpolteedintahnedtsoeparrcehseudltsfoarnitds /article
tureofthearticleisthefollowing.First,weanalyzethedeep occurrencesasfortheotherprofiles.Webuiltawebserverto -ab
s
branching of the key proteins that have homologs among allowrunningtheproteinprofiles.Thisisavailableathttp:// tra
(nearly) all conjugative systems of a given kind. This allows mobyle.pasteur.fr/cgi-bin/portal.py#forms::CONJscan- ct/3
uncoveringtheinitialsplitoftheproteinsthatbecamekeyto T4SSscan. 0/2
conjugativeprocesses.Then,wefocusontheearlyeventsof /3
1
5
the diversification of ssDNA conjugation, by far the most Phylogenetic Analysis /10
frequentprocessamongprokaryotes.Finally,wedetailthedi- 1
3
versification of the best-known conjugation families within Unless explicitly stated, all phylogenetic analyses were per- 98
ssDNA-basedsystemswithafocusontheevolutionofgene formed with the following procedure. First, sequences 1 b
y
repertoires and MOBless T4SS. This analysis provides infor- were aligned using MUSCLE with default parameters as g
u
implementedinSeaView (Gouyetal.2010).Second,allcol- e
mationthatnaturallyleadstoarevisionofT4SSclassification s
basedonevolutionarybiology. umnsinthemultiplealignmentmatrixwithmorethan80% t on
of gaps were removed. Third, 100 replicate trees were built 1
1
Materials and Methods with RAxML 7.2.7 (Stamatakis 2006) using the model A
p
Data GTRGAMMA. We kept the one with the best likelihood. ril 2
Wecalculatedbootstrapswiththestandardimplementation 01
Dataoncompletechromosomesandplasmidsofprokaryotes 9
and used the autoMR stop criterion to obtain confidence
were taken from Genbank Refseq (ftp://ftp.ncbi.nih.gov/gen
values for each node. There were two exceptions to this
omes/Bacteria/,lastaccessedNovember2011).Thisincluded
method. We aligned the ATPases using MAFFT (Katoh and
1,207chromosomes,891plasmidsthatweresequencedalong
Toh2010)withtheG-INSIalgorithmandremovedthesites
with these chromosomes, and 1,391 plasmids that were
containingmorethan60%ofgaps.Weperformedthephylo-
sequenced independently. We used the annotations of the
geneticinferenceasmentionedearlierandadditionallywith
Genbankfiles,havingremovedallpseudogenesandproteins
PhyML3.0(Gascueletal.2010)undertheLGmodelandwith
withinnerstopcodons.TheinformationonT4SSwastaken
thebioNJstartingtreetogetaLRTsupportvalues.Thealign-
fromGuglielminietal.(2011).
ment of the set of VirB4 and VirD4 was built with MAFFT
with the E-INSI algorithm, since these two proteins show
Construction of Protein Profiles and Genome Searches different domain organization, and then manually edited.
Unless mentioned explicitly, the protein profiles used are MAFFT was used instead of MUSCLE because it provided
those described in Guglielmini et al. (2011). To study the better alignments in these cases. The computation of
317
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Guglielminietal. . doi:10.1093/molbev/mss221
100 replicates plus hundreds of bootstrap trees was exces- RelativeDecreaseinProteinSimilaritywithDivergence
sively time consuming, given the size of the data set in the For each pair of T4SS loci, we made pairwise alignments of
VirB4/VirD4 analysis. Thus, we used PhyML 3.0 to build the eachoftheorthologouspairsofgenes.Alignmentsweredone
phylogenetic tree, under the LG model and with the bioNJ using an end-gap free version of the Needleman–Wunsch
starting tree. aLRT support values were also calculated for algorithm (Mount 2004), with a BLOSUM60 matrix, open
eachnode. penaltyof1.2,andextensionpenaltyof0.8.Wethenplotted
The support tests we conducted revealed in this last the percentage of similarity between VirB4 homologs and
tree some weak support that conflict with the aLRT values. each of the other pairs of homologs. The points for each
To further investigate this, we used a reduced data set scatter plot were then fitted with a spline (l=1,500), and
composedofVirB4proteins,excludingthedistanthomolog thecurvesweresuperimposed.
TraU. Using this data set, we performed the tests
Results and Discussion
described later. All multiple alignments and phylogenetic
reconstructions are freely available on DRYAD (http://data Early Evolutionary Split of the Key Conjugation ATPases D
o
dryad.org/). w
The two families of T4CPs (with prototypes given by the n
lo
VirD4pTi and TcpApCW3), the two families of ATPases ade
(basedonVirB4 andTraU ),thedsDNAconjugationpro- d
TToesttessttothteheroPbhuystlongesesnoetficouArncaolnycsilussions based on phylo- teinTraBpSG5,anTdi FtsKareaRll6p4artofthesuperfamilyofAAA+ from
motor ATPases. Hence, we investigated the events at the h
geneticanalysis,wemadeanumberof tests.Theseanalyses ttp
onsetofthenaturalhistoryofconjugationfromtheanalysis s
aimed at testing the robustness of the conclusions to the ofthephylogeny-linkinghomologsforalltheseproteinpro- ://a
multiple alignments, to the identification of informative ca
filesamong3,489replicons(seeMaterialsandMethods).The d
sites in multiple alignments, and to the use of a protein tree was rooted using the distantly related protein family em
modelmatrix.Wethereforeproducedtwoautomaticmeth- derived from VirB11 (Planet et al. 2001). The monophyly ic.o
ods where we make the alignment of the protein using ofVirB11isrobustinTbiothexpertandtheautomaticanalyses up.c
MAFFTandMUSCLE.Informativesiteswereextractedfrom o
(table1).Thisphylogeneticreconstructionseparatesamono- m
the alignments using BMGE (Criscuolo and Gribaldo 2010). /m
phyletic VirD4/VirB4 clade (67% boostrap) from the others.
b
We fine-tuned BMGE parameters for each alignment to e
Thisfitspreviousgenomicandstructuralanalysisshowingthe /a
obtain a good compromise between the quality and the similaritybetweenthedsDNAtranslocatorsFtsKandTraBon rtic
number of informative sites. The best model to analyze the le
the one hand and between the ssDNA translocators VirD4 -a
datawaschosenwithProtTest(Darribaetal.2011).Notethat b
andVirB4ontheother(Iyeretal.2004;Cabezonetal.2011). s
ProtTestdoesnotanalyzetheGTRmodelforproteins,sowe tra
Thepreviousanalysisallowsrootingthetreeandhighlights c
cannotassesswhetherthemodelchosenbyProtTestisbetter theearlysplitbetweenssDNAanddsDNAtranslocases.But t/30
than ours. Trees were built as before using RAxML, and we /2
theinclusionofthedistantlyrelatedVirB11producesamul- /3
generated100bootstraptreesforeachanalysis.Tocompare 1
tiple alignment with few sufficiently conserved positions, 5
the different analyses, we computed the quality of multiple increasing uncertainty in the process of phylogenetic infer- /10
1
alignment score using the Core component of T-Coffee 3
ence (supplementary table S1, Supplementary Material 9
8
(Notredame et al. 2000) for the three methods (our expert online). This reduced the power of this data set to robustly 1 b
analysis,theMAFFTandMUSCLE-basedanalyses).Thisscore, resolvethemorerecentsplits.Thus,weexcludedVirB11from y g
ranging from 0 to 100, is computed by comparing the con- theanalysisandmadeanewphylogeneticreconstructionof ue
s
sistencyofthealignmentwithalistofprecomputedpairwise theremainingfivefamilies.Thistreeshowsthesamedichot- t o
n
alignmentscalledlibrary.Weusedthedefault“Mproba_pair” omyatthebase(fig.2),withstrongsupportforallfivemono- 1
1
library. The key results, e.g., monophyly or basal position of phyletic groups with our expert analysis and in the best A
p
certain clades, were tested for the three methods and are automaticmethod(table1).Theseresultsfitourobservation ril 2
displayedintable1andsupplementarytableS1,Supplemen- that our VirB4 protein profiles often match VirD4 proteins 01
9
taryMaterialonline.Eachofthesetestshasanidentification andviceversa,albeitwithweakscores,andthatnoneofthese
numberinthetables.Thisnumberisdisplayedintherespect- match significantly proteins from the families TraB/FtsK.
ive node in the phylogenetic trees. For example, in figure 2, T4CPs and VirB4s show clear structural similarities, under-
thenodewithIDno.3referstothemonophylyofTraBandis scoring a common functional mechanism (Cabezon et al.
indicatedintable1ashaving99%bootstrapsupportinour 2011). The most conspicuous structural difference between
expertanalysis,100%intheautomaticanalysisusingMAFFT, T4CPsandVirB4sistheexistenceofthreealphahelicesthat
and96%intheautomaticanalysisusingMUSCLE.Insupple- are conserved in the C terminus of VirB4 proteins but are
mentary table S1, Supplementary Material online, it is indi- absent in T4CPs. Deletion of these helical structures in the
cated that for this analysis the best alignment, as given by VirB4 homolog TrwK resulted in a large increase in its
R388
T-Coffee, is the one of the expert alignment (score 88), fol- ATPase activity, suggesting that the C-terminal end of
lowedbyMAFFT(76)andthenMUSCLE(67).Thenodeno.3 VirB4 proteins functions as an autoregulatory element
infigure2isthusindicatedinablackcircle(highbootstrap (Penaetal.2011).Overall,theseanalysesfitstructuralwork,
support). suggesting that the common ancestor of the VirB4/VirD4
318
MBE
EvolutionofConjugationandT4SS . doi:10.1093/molbev/mss221
U plit
a s
OtherInformativeTraits Essentialubiquitousgeneinbacteria TenspecificgenesforthisMPF Fourspecificgenes FivespecificgenesNinespecificgenesforthisMPF SpecificityofprimarysequenceofTr Geneorderanalysisshowsthesame UseofTcpAandMOBT ber.
m
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USCLEBMGE 871 19969569 1009489999685942318 10074553318 10057 77 17951009700 52245249197 urewit nload
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otstrapValu +MAFFTBMGE 9630 821009938 100956210010053874537 10097673336 10090 100 29911009900 999890211100 nthecorrespo https://aca
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OriginalAlignment 10067 78999662 1009899100100100982633 10098 100 21961009600 87791007985100 displayedhere ic.oup.com/m
is b
cNumber 1 2345 6791011121314 78151617 1819 20 212223 242526 2728 greaterthan50,it e/article-abstra
conservation supportvalueis ct/30/2/315/10
order ifthe 1398
ne and 1 b
Table1.AnalysisoftheRobustnessofKeyPhylogeneticResults. abDataSetHypotheses +ATPasesVirB11VirB11monophylyTraB,TcpA,FtsKmonophyly ATPaseswithoutVirB11VirB4,VirD4monophylyTraBmonophylyTcpAmonophylyFtsKmonophyly VirB4,VirD4MPFmonophylyImonophylyMPFFAmonophylyMPFTmonophylyMPFCmonophylyMPFGmonophylyMPFFmonophylyMPFBBasalMPFIBasalMPFC VirB4(withoutMPF)MPFmonophylyIFAmonophylyMPFFATAMonodermsmonophyly,MPFmonophylyMPFFB,MPF,MonodermsmonophylyMPFFB VirB4(MPF)SamebasalcladeinallanalysesTTwobigcladescorrespondingtothege VirB4(MPF)SamebasalcladeinallanalysesF VirB4(MPF)ArchaeamonophylyFATAEuryarchaeotamonophylyCrenarchaeotamonophylyTenericutesmonophylyActinobacteriamonophylyFirmicutesmonophyly )FirmicutesImonophylyVirB4(MPFFAFirmicutesIImonophylyBasalFirmicutesIIIActinobacteriamonophylyActinobacteriaImonophylyActinobacteriaIImonophyly aProteinsincludedinthedataset.bThedifferenthypothesesforwhichwepresentthebootstrapsupports.cWhenthehypothesiscorrespondtowhatweobserveinthereferencephylogeny,dBootstrapvaluesforeachhypothesisandforeachalignmenttechnique. y guest on 11 April 2019
319
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Guglielminietal. . doi:10.1093/molbev/mss221
Acidobacteria
Proteobacteria
Planctomycetes
0.5 Fusobacteria
Deinococcus-Thermus
Bacteroidetes
Chlorobi
5 Firmicutes FtsK
Actinobacteria
Chloroflexi
Chlamydiae
Archaea
Cyanobacteria
3 TraB
(Actinobacteria)
1
D
o
w
n
lo
a
d
e
4 T(FciprmAicutes) d from
h
ttp
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FIG.2. PhylogeneticanalysisoftheAAA+ATPasesassociatedwithconjugation.ThepositionoftherootwasdeterminedusingtheAAA+ATPase t/3
0
VirB11inaseparateanalysis.NamesalongtheFtsKtipscorrespondtothetaxonomicoriginsofeachprotein,reflectingthewidthofsampling.Bold /2
verticalblacklinesrepresentnodeswithahighsupportvalue(bootstrap>70%andaLRT>0.7).BoldgraylinesrepresentnodeswithhighaLRTscore /31
5
(>0.7)butaweakerbootstrap(<70%).ThehomologsofTcpAarefoundonlyinFirmicutes.ThehomologsofTraBarefoundonlyinActinobacteria. /1
0
Numbersincirclesrefertotheanalysisofrobustnessintable1(identifiedinthethirdcolumnoftable1);blackbackgroundstandsforahighsupport 1
3
((cid:2)70%bootstrapinthebest-scoringalignment)andgraybackgroundforamoderatesupport((cid:2)50%bootstrapinthebest-scoringalignment). 9
8
1
b
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familiesconsistedofasolubleproteinengagedinpolypeptide Actinobacteria and are related with FtsK, but they do not g
u
transport(asit’sstillthecaseinmoststudiedVirB4proteins). emergefromwithintheFtsK.Instead,theyderiveindepend- es
VirB4laterbecamemembraneboundbyassociationwiththe ently from the ancestor of this protein. FtsK is an essential t o
n
cVthairanBtb3sepcceoocmivaaplilzoeenndten(iantssoisnfDVTN4irASBS4tRr(6aaKns)si(pnPoeVrntira(BTe4t4RCa38lP.82)).0aT1ls1ho)is.aTachsqesuopicrrieoadttieoainnn p2(Y0roP0t_4e)5i,n03itn3h0ca7ltu.,1dc)eo.snTthraaetrylaltetotaessrtomisoenanpenreomvtiaoetmuesdbseuargsgFaetmsstKioo-nnligkse(IAyperrrcohetteaieanal,. 11 April 2
0
integral-membrane protein domain in its N-terminus. This anditisnotcloselyrelatedwithHerAproteins,whichbranch 1
9
component is involved in its interaction with another T4SS closertotheVirD4/B4branches,anditsstudyfallsoutsidethe
component,inthiscaseVirB10(Llosaetal.2003;dePazetal. scope of this article. FtsK phylogeny follows approximately
2010). theoneofbacteria(Gupta2004)andthusprovidesaguide-
The other basal branch in the phylogeny includes TraB, line to the timing of the diversification of these protein
TcpA, and FtsK, all with strong to moderate evidence of families. The tree in figure 2 shows that proteins have
monophyly (99%, 96%, and 62% bootstraps, respectively) widely diverse tip-to-root branch lengths, i.e., the proteins
(fig. 2). The relative order of the split between the three donotevolveaccordingtoastrictmolecularclock.Therefore,
clades is different from a previously published one, but its wecannotassumeamolecularclockthatwouldallowdating
bootstrapsupportisweakinourtree(andnotdocumented the split of these families and thus presumably that of
inParsonsetal.2007).SpoIIIE,aproteininvolvedinsegrega- conjugation processes. Yet, this data does place the origin
tionofchromosomesduringBacillussubtilissporulation(Wu of ssDNA conjugation extremely early in the history of
andErrington1998),brancheswithintheFtsKclade(datanot life. While TraB and TcpA seem to diversify after FtsK, in
shown). The elements of the TraB family are found only in agreement with their presence only in Firmicutes and
320
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EvolutionofConjugationandT4SS . doi:10.1093/molbev/mss221
Actinobacteria, the diversification of the pair VirB4/VirD4 only in Proteobacteria, and its features are largely unknown
could be contemporaneous or shortly subsequent to that (Juhas,Crook,etal.2007;Juhas,Power,etal.2007).Interest-
ofFtsK.Theseresultssuggestthatthetwoconjugationmech- ingly, an MPF encoding element, the PAPI-1 pathogenicity
G
anisms, ssDNA and dsDNA conjugation, are based on islandofPseudomonasaeruginosa,hasseveralgeneshomolo-
ATPases that diverged before the last common ancestor of goustothethinpilusofR64(Carteretal.2010).Hence,the
bacteria. associationbetweenMPFandthinpilimightbeanancestral
trait.
Four groups correspond to the different T4SS families of
T4SS Phylogeny
Proteobacteria(MPF ,MPF ,MPF,MPF )(Juhas,Crook,etal.
WealignedtheproteinsmatchingtheVirB4andTraUprofiles F G I T
2007;Smillieetal.2010).Thesefourgroupsareclearlysepa-
to infer the evolutionary history of all VirB4 homologs. We
ratedbecausetheyallhavestrongbootstrapsintheanalysisof
then used VirD4 to root this tree. Despite relatively weak
monophyly(table1),andeachcontainsasetoffourtonine
support in the bootstrap tests (48% in the best automatic
genesthatarespecific,i.e.,theirproteinprofilesmatchlociof
D
alignmentand69%inourexpertanalysis),thisrootingshows a given MPF but not those of the other MPF types (Smillie ow
agoodaLRTsupportvalue(0.82),consistentwiththelitera- n
etal.2010).Interestingly,307outof327(94%)oftheT4SSof lo
ture in terms of phylogeny and biochemical function (Iyer a
Proteobacteria are classed in one of these four clades. We d
e
etal.2004;delaCruzetal.2010;Smillieetal.2010)andwith d
thepreviousanalysisofthefiveATPases(78%boostrap).The investigated the loci of the 20 remaining VirB4 proteins. fro
Oneofthemdoesnotcolocalizewithanyoftheothercon- m
tcwrlaeesells-issfiuhepodpwoisnrtttoehdaeticgalhalldteVgsirroB(ufi4gp.as,n3wd).hTTichraheUat-wrreeolaretbepadrseapslergnorttoeeuidnpsbsycianenigthbheet jooutnghlayetroion1ne9,pMVriorPBtFe4itnyaprpeer.oeTfinhlceeosy,dwiendecrlenuendaionrtggcelraneslesasexdasspaeesscaiafigncivdeonTf4oMCnPPeF., Tajunhsdet https://ac
a
VirB4 phylogenetic reconstruction are MPFI followed by a because the number of these specific genes is below the de
groupspecifictoCyanobacteria(MPF ).Thisisinagreement m
C quorum we set up as a minimum for a putative complete ic
with the low similarity between TraUR64 (MPFI) and VirB4Ti T4SS (Guglielmini et al. 2011). Many of these 20 unclassed .ou
(MPFT) that had prevented previous phylogenetic recon- elementsarethusprobablyinactive,enduringageneticdeg- p.c
o
structions of all VirB4 homologs (Smillie et al. 2010). With radation that results in incomplete loci. Alternatively, they m
theavailabilityofmoresequencesoftheseproteins,notably may correspond to highly modified versions of T4SS; the /mb
e
cyanobacteria,andtheinclusionoftheT4CP,wecouldnow H. pylori Cag-pathogenicity island is notably found within /a
reconstruct a reliable phylogeny. However, the position of theseelements. rtic
le
MPFI at the basis of the tree must be taken with care. Our Afew genomes of species not classedamong Proteobac- -a
b
expert method and the two controls produce MPFI at the teria encode T4SS classed within MPFF and MPFT. All these stra
bbaosoistsotrfatpheinphthyelogbeenstyabuuttowmiathticrealalitginvemlyenlotw) (stuapbpleor1t).(4T5h%e boauctteerrimaeamrebdraidneer.mTsh,isi.el.i,stthienycluhdavees bMoOthBlaenssinTn4eSrSainndoanne ct/30
/2
MPFC clade often arises at the basis in the bootstrap trees Aquificae(MPFF)andoneProtochlamydia(MPFF),andcon- /31
orasasistercladeofMPFI.Inanycase,thisanalysisplacesone jugative T4SS in one Chlorobi (MPFT), one Deferribacteres 5/1
ofthesetwocladesattherootofthetreeinmorethan85%of (MPFF), one Acidobacteria (MPFT), and two Fusobacteria 013
theboostrapanalyses. (MPF ). These elements are scattered in the trees of MPF 9
T T 8
Some mobile elements encoding an MPFI, e.g., the R64 andMPFF(figs.4and5),suggestingdifferenteventsofhori- 1 by
plasmid from the MOBP12 family, besides encoding a thick zontaltransferfromProteobacteria.Indeed,theydonotclus- gu
rigidpilus,withhomologytoMPFT,alsoencodeathinpilus tertogetherinthephylogenetictrees(0%inbootstraptrees). es
that is only required for conjugation in liquid and that is Theelementsofeachgivenbacterialcladearealwaysmono- t o
n
homologous to type IV pili (Kim and Komano 1997). This phyletic, suggesting one single transfer event, but the very 11
led to the classification of MPFI as T4SSb in opposition to small number of such elements does not allow any robust Ap
MPFF and MPFT, both classed as T4SSa (Christie and Vogel conclusions for the moment. Only one nonproteobacterial ril 2
2000).However,otherMPFIelements,e.g.,plasmidCTX-M3, clade,Acidobacteria,isbasalinthetreeofMPFT(100%boot- 019
lack a thin pilus and are still able to mate in liquid at high strapsintheexpertanalysisandthecontrols).Acidobacteria
frequency (Golebiewski et al. 2007). Thus, the thin pilus of areoftenregardedasasistercladeofProteobacteria(Ciccar-
MOB plasmidsisjustanadditionalfeatureofsomeMPF elli et al. 2006), and therefore, we cannot discard the possi-
P12 I
systems,actingprobablyjustasafacilitatorofliquidmating bility of a diversification of MPF before the split between
T
and a selector of recipients (Kim and Komano 1997), while AcidobacteriaandProteobacteria.However,sinceMPF and
G
thecoreMPF machineryformsthebasisofthisconjugation MPF aremorebasalinthetreeofVirB4(fig.3),andbothonly
I I
system.Inanycase,thehighlydivergentnatureofTraU isa foundinProteobacteria,thescenarioofatransferfromPro-
R64
signature for this whole family of liquid maters. Nothing is teobacteria to Acidobacteria remains more parsimonious.
known experimentally about MPF . Because cyanobacteria Interestingly,allT4SSpredictedinthesesixnonproteobacter-
C
divergedearlyonfromProteobacteria,MPF mightalsocon- ial clades were classed among MPF and MPF . Nothing is
C F T
tainpeculiaritiesrelevanttothegeneticorphysicalenviron- knownaboutconjugationintheseclades,butthisdatasug-
mentoftheseorganisms.MPF isthenextmostbasalgroup gest they might use mechanisms closely related to, and ori-
G
inthetree.Thissystemwasrecentlydiscovered,wasidentified ginatingfrom,thoseofProteobacteria.
321
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Guglielminietal. . doi:10.1093/molbev/mss221
MPF
FATA
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7 MPF
FA
MPF
1122 F
MPF
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FIG.3. JointphylogeneticreconstructionoftheVirD4andVirB4/TraUfamiliesofproteinsfromconjugativesystems.Boldverticalblacklinesrepresent t o
n
nodeswithahighsupportvalue(aLRT(cid:2)0.9),andblackverticalgraylinesrepresentnodeswithasupportvaluebetween0.7and0.9.Blacksquare 1
1
bracketsindicatetheVirB4andVirD4clades;coloredsquarebracketsontheleftdelimitthedifferentMPFclades(purple:MPFFATA,orange:MPFFA,red: A
p
MpaPrtFFo,fbtlahcekt:rMeePF(bB,lubelu:eM:MOBPPF,Tg,ryeeellno:wM:MOPBFQG,,rceyda:nM:MOBPFF,Cp,gurrepelen::MMOPFBI)B;,coorlaonregde:sMquOaBreHb,brarcokwentsMonOtBhCe,rriegdh/tgdreeelinmditatshheedreblarxaacskeectsla:dcleasdwesitwhiinththaemViixrDo4f ril 20
1
MOB andMOB ;black:mixofMOB,MOB andMOB ).Numbersincirclesrefertotheanalysisofrobustnessintable1(identifiedinthethird 9
F Q P F H
columnoftable1);blackbackgroundstandsforahighsupport((cid:2)70%bootstrapinthebest-scoringalignment)andgraybackgroundforamoderate
support((cid:2)50%bootstrapinthebest-scoringalignment).
Phylogeny of the T4CP at the Light of VirB4
MPF ). VirD4 of the two remaining clades (MPF and
C T
Phylogeny MPF ) are scattered in a small number of clades. Most of
F
ThetreesofVirD4andVirB4arenotcongruent(ELWconfi- the MPF use TcpA instead of a VirD4-like T4CP (see
FA
dencevalue:0,andSHPvalue<0.01).Yet,theysharemany later).ThefewVirD4proteinsfoundinMPF arealsomono-
FA
features (fig. 3). The proteins encoded by the virD4 genes phyletic (orange in the bottom of fig. 3). It was previously
colocalizing in replicons with virB4 tend to form similar shownthatplasmidT4CParesometimesscatteredindiffer-
clades.Notably,theVirD4associatedwitheachofsixofthe ent groups corresponding to given relaxases (Smillie et al.
eight VirB4 clades also clustered in nearly monophyletic 2010). This result is still valid with the present much larger
clades of T4CP (MPF , MPF , MPF , MPF , MPF, and dataset.Forexample,theT4CPcladewithamixtureofMPF
FA FATA B G I T
322
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EvolutionofConjugationandT4SS . doi:10.1093/molbev/mss221
pCRY
R6K
Brucella VirB T4SS
Bartonella Trw T4SS
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FIG.4. PhylogeneticanalysisofMPFTVirB4proteins.Boldverticalblacklinesrepresentnodeswithahighsupportvalue(bootstrap>90%),andbold 013
verticalgraylinesrepresentnodeswithasupportvaluebetween70%and90%.GreenbranchescorrespondtotaxathatarenotwithinProteobacteria 9
8
(or the outgroup). Red branches represent VirB4 not associated to a relaxase (MOBless T4SS). The leftmost vertical bar on the right stands for 1 b
chromosomal (black) or plasmidic (white) proteins. The colored bar represents the different gene order patterns found; the patterns and their y g
correspondingcoloraredepictedatthebottom(thenumbersrepresentthecorrespondingvirBgene);apatternisattributedtoasystemif,considering ue
s
thepossiblymissingvirgenes,thegeneorderispreserved.Forexample,asystemcomposedofthegenesvirB1,virB4,virB6,virB5,virB8,virB9,andvirB10 t o
inthisorderwillbeassignedtotheorangepattern.Uniqueoratypicalpatternsaredepictedinblack.Knownrepresentativesystemsarelabeled. n 1
Numbersincirclesrefertotheanalysisofrobustnessintable1(identifiedinthethirdcolumnoftable1);blackbackgroundstandsforahighsupport 1 A
((cid:2)70%bootstrapinthebest-scoringalignment)andgraybackgroundforamoderatesupport((cid:2)50%bootstrapinthebest-scoringalignment). pril 2
0
1
9
andMPF hasonetypeofrelaxaseincommon(MOB ).On membranes, i.e., diderms (98–100% of the bootstrap trees
F F
the other hand, some relaxase types are scattered among inallthreeanalyses).ThisstronglysuggeststhatssDNAcon-
differentVirD4cladesthat follow MPF types, e.g.,the VirD4 jugation was invented among diderms. In this scenario,
associated with MPF is monophyletic and includes three ssDNAconjugationwouldhavebeenacquiredbymonoderm
C
differentrelaxases,whicharealsofoundinotherMPFtypes. prokaryotes,i.e.,organismsdevoidofanoutermembrane,by
Hence,evolutionofconjugationisdrivenbytwomaincon- HGT.Thisalsofitstheobservation thatallmonoderm con-
straints,oneactingmainlyontheT4SS,representedbyVirB4, jugationsystemsareintwosisterclades:MPF andMPF
FA FATA
and other on the relaxosome, represented by the relaxases. (monophyleticin67–55%ofthebootstraptrees).
T4CPtendstocoevolvewithbothcomponents. MPF includessixdistinctgroupsofFirmicutes(mono-
FATA
phylyofallFirmicutessupportedby0%ofthebootstraptrees,
Cell Envelope Adaptation in Monoderms table1),twoofActinobacteria(monophylyofallActinobac-
The most basal clades in both VirB4 and VirD4 phylogenies teria supported by 0% bootstrap trees), one of Tenericutes
correspond to bacteria with both inner and outer (monophyly of the clade supported by 96–99% bootstrap
323
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Guglielminietal. . doi:10.1093/molbev/mss221
plasmid R100
F plasmid
L. pneumophila pLPL D
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FIG.5. PhylogeneticanalysisofMPFFVirB4proteins.Boldverticalblacklinesrepresentnodeswithahighsupportvalue(bootstrap>90%),andbold 15
verticalgraylinesrepresentnodeswithasupportvaluebetween70%and90%.GreenbranchescorrespondtotaxathatarenotfromProteobacteria /1
0
(plustheoutgroup).RedbranchesrepresenttheVirB4notassociatedtoarelaxase(MOBlessT4SS).GreenandreddottedbranchesrepresentMOBless 1
3
9
T4SSthatarenotfromProteobacteria.Thebarontherightstandsforthechromosomal(black)orplasmidic(white)proteins.Knownrepresentative 8
1
systemsarelabeled.TheGGIDNAreleasesystemcorrespondstotheN.gonorrhoeaegonococcalgeneticisland(Hamiltonetal.2005).Numberincircles b
y
referstotheanalysisofrobustnessintable1(identifiedinthethirdcolumnoftable1);blackbackgroundstandsforahighsupport((cid:2)70%bootstrapin g
u
thebest-scoringalignment)andgraybackgroundforamoderatesupport((cid:2)50%bootstrapinthebest-scoringalignment). e
s
t o
n
1
1
trees), and a group of Archaea unlikely to be monophyletic contributedtothescattereddistributionoftaxainthephylo- A
p
(bootstrapofonly17–29%)withaclearseparationbetween genetictreeofMPFFATAandMPFFA. ril 2
EuryarchaeotaandCrenarchaeota(91–96%,respectively,and The MPFFA clade includes two groups of Actinobacteria 019
100% bootstrap support for each clade) (fig. 6). The deeper intermingledwiththreegroupsofFirmicutes(<5%bootstrap
relations between these clades are difficult to disentangle, support for a net separation of the two clades) (fig. 7). The
given the low bootstrap supports of the basal nodes. mostbasalgroup(FirmicutesIIIinfig.7)isconstitutedbya
Within the Firmicutes clades, we find the main divisions, few elements from Firmicutes (bootstrap support for this
i.e., Bacillales, Lactobacillales, and Clostridia, scattered in the basal position of 52–100%, table 1). This suggests that the
tree. This suggests that, once a conjugative system arose in ancestral conjugative system might have arisen within
thisphylum, itspreads early among the main divisions, and Firmicutes from which it was transferred to Actinobacteria.
transfersbetweendivergentcladesweremaintainedthrough This is consistent with the observation of a basal group,
a certain moment in evolution. The monophyly of mono- including only Firmicutes and Tenericutes in the sister
derms in the VirB4 tree suggests that monoderms acquired MPF tree (fig. 6). The subsequent split in the MPF
FATA FA
conjugative systems by transfer from diderms. This early group separates a clade with Actinobacteria and Firmicutes
acquisition was followed by the adaptation of the T4SS to II from Firmicutes I (fig. 7). The latter encodes TcpA as a
monoderms. Finally, frequent conjugation between diderms putative T4CP, which further supports the monophyly of
324