Coupling Factors in Macromolecular Type-IV Secretion Machineries

Coupling Factors in Macromolecular Type-IV Secretion Machineries Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1551

Coupling Factors in Macromolecular Type-IV Secretion Machineries

F. X. Gomis-Rüth1,*, M. Solà1, F. de la Cruz2 and M. Coll1,*

1Institut de Biologia Molecular de Barcelona, C.S.I.C., c/ Jordi Girona, 18-26, E-08034 Barcelona (Spain) and2Departamento de Biología Molecular (Laboratorio asociado al C.I.B., C.S.I.C.), Universidad de Cantabria, c/ HerreraOria, s/n, E-39011 Santander (Spain)

Abstract: Type IV secretion systems (T4SSs) are bacterial multiprotein organelles specialised in the transfer of(nucleo)protein complexes across cell membranes. They are essential for conjugation, bacterial-induced tumour formationin plant cells, as observed in Agrobacterium, toxin secretion, like in Bordetella and Helicobacter, cell-to-cell translocationof virulence factors, and intracellular activity of mammalian pathogens like Legionella. By enabling conjugative DNAdelivery, these systems contribute to the spread of antibiotic resistance genes among bacteria. These translocons are madeup by 10-15 proteins that are analogous to Vir proteins of Agrobacterium and traverse both membranes and theperiplasmic space in between in Gram-negative bacteria. Their secretion substrates range from single-stranded DNA /protein complexes to multicomponent toxins and they are assisted by integral inner-membrane coupling factors, themultimeric type-IV coupling proteins (T4CPs), to connect the macromolecular complexes to be transferred with thesecretory conduit. To do so, these T4CPs may be required to localise close to the secretion machinery within the donorcell. The T4CP structural prototype is the hexameric protein TrwB of Escherichia coli conjugative plasmid R388, closelyrelated to Agrobacterium VirD4 protein. It is responsible for coupling the relaxosome with the DNA transport apparatusduring cell mating. T4CP family members are related to SpoIIIE/FtsK proteins, essential for DNA pumping duringsporulation and cell division. These features suggest possible mechanisms for conjugal T4CP function: as a simplecoupler between two molecular machines, as a rotating device to pump DNA through the type-IV transport pore, or as aDNA injector, whereby its central channel would function as part of the transport pore.

Key Words: bacterial conjugation, coupling protein, type-IV secretion systems, DNA transfer, helicase, three-dimensionalstructure.

TYPE-IV SECRETION, BACTERIAL CONJUGATIONAND PATHOGENESIS

Bacteria are not solely dependent on their internal milieuto accomplish their living strategies. They often secreteenzymes and other macromolecules to the external mediumin order to readapt it according to their own requirements.Secretion machineries spanning the cell membrane(s) areresponsible for macromolecular transport. These systems canbe classified into five types (I to V) and share the presence ofproteins using nucleosyl triphosphate (NTP) hydrolysis as atransport driving force, except for the autotransportersystem, which does not depend on NTP [1-10]. Over a dozendistinct families of protein translocating systems, mostlyrestricted to bacteria, have been characterised and classified[11, 12]. A sophisticated variant of macromolecular secretionis type-IV translocation, through which (nucleo)proteins canbe directly injected into prokaryotic and eukaryotic recipientcells. In an extreme version, DNA is translocated to therecipient cell, so that its metabolism is permanently altered.The type-IV pathway encompasses a multiprotein bacterialtransmembrane organelle or translocon specialised in theintercellular transfer of a variety of macromolecules. Itfeatures a filamentous macromolecular apparatus protruding

*Address correspondence to these athors at the Institut de BiologiaMolecular de Barcelona, C.S.I.C., c/ Jordi Girona, 18-26, E-08034Barcelona (Spain); Tel: +3493 400 61 44/49; Fax: +3493 204 59 04; E-mail:[email protected] and [email protected] the bacterial envelope. T4SSs can be controlled bytranscriptional repressor systems, like KorA/KorB, which

regulate partitioning genes [13]. It differs from the other foursystems in that it is conjugation-related or an “adaptedconjugation” system, capable of DNA translocation [11, 14,15]. The substrates are as diverse as nucleoprotein particlesand multicomponent protein associations that cross thebacterial envelope into bacterial, plant and mammalian cells,i.e. even across kingdom boundaries, and into the extra-cellular space [15-17]. The engagement of secreted substratescan also occur in the cytoplasm, the inner membrane or theperiplasm of the bacterial cell [1]. These transloconstypically consist of 10-15 proteins, encoded by a single genecluster, and they form two major structural components: anextracellular filamentous surface appendage or sex pilus, forinitiating physical contact between the donor and recipientcells [18], and a membrane-associated protein complex thattranslocates substrates across the membranes involved [19].It is still a matter of debate whether the pilus belongs to theconjugative pore or merely brings cells together. Since thepilus is not present in Gram-positive bacteria, it does notseem to be indispensable.

This pathway is involved in bacterial conjugation, acontact-dependent process often induced by extracellular orgrowth-phase dependent signals [20-23]. It is mediated byconjugative plasmids or transposons that transfer DNAunidirectionally from a donor to a recipient cell across thedonor cell envelope, rendering a recipient that may become apotential donor (Fig. (1a); [21, 24]). It is a means for rapidevolution, by which bacterial populations adapt to achanging environment, e.g. acquisition of resistance toantibiotics. It is also a broadly applicable tool to shuttle

1552 Current Pharmaceutical Design, 2004, Vol. 10, No. 13 Gomis-Rüth et al.

genes between different species and even for trans-kingdomgene transfer [25-30]. Conjugative DNA transfer depends ongenes contained in the plasmid transfer (tra) region, furtherdivisible into dtr (from DNA transfer and replication) andmpf (from mating-pair formation; [26]). Dtr proteins processconjugative DNA through formation of the relaxosome, anucleoprotein complex. This implies sequence-specificrecognition of a cis-acting DNA sequence called origin oftransfer (oriT) and a DNA site- and strand-specific cleavageby specialised nicking enzymes, or relaxases, that hydrolysea single phosphodiester bond and attach covalently to thenewly created 5’ terminus. This process is followed by astrand displacement reaction, which generates a single-stranded (ss) DNA transfer intermediate, the T-strand [21]. Asimultaneous rolling-circle DNA replication reaction isobserved for replacement of the displaced strand in the donorcell (Fig. (1a); [30]). The relaxosome is attached to thetransport site facing the cytoplasmic side of the cell envelope[31] and the DNA-transfer channel complex, a T4SS formedby 11-12 plasmid Mpf proteins [26], transports the T-strandto the recipient cell with 5’ to 3’ polarity. Conjugativesystems, an example is Escherichia coli F sex factor conju-gation [32], may also deliver non-self-transmissible plasmidsto recipient cells [25, 33] and translocate DNA even acrosskingdom barriers, e.g. to Saccharomyces cerevisiae [29].Furthermore, these systems may shuttle proteins to recipientcells independent of DNA, as shown for RecA protein duringthe transfer of plasmid RP4 to E. coli recipients [34].

In the soil phytopathogen Agrobacterium tumefaciens,the type-IV VirB transporter translocates a segment of thetumour-inducing 200-kb Ti plasmid DNA, the T-region, inthe form of an ss nucleoprotein T-complex with thecovalently attached VirD2 protein from the bacterium todicotyledonous plant cells. This results in phytohormoneoverproduction and crown-gall disease in the latter [1, 35-38]. This translocon has been exploited in plant biotech-nology as a shuttle to introduce new genes conferringresistance to herbicides and pathogens in industrial crops[39]. Transfer is mediated by a set of 20 virulence (Vir)proteins encoded by the Ti plasmid, 11 of which assemble asa transporter [20]. The process requires additional proteintranslocation into the plant cell, so as precise cleavage andcyclisation of pilin-like protein VirB2 [35, 40]. The VirBsystem also mediates a conjugation-like transfer of suitableplasmids to recipient plant and bacterial cells [25, 37] and ofT-DNA to the three major groups of fungi, the molds, theyeasts, and the mushrooms, as shown for S. cerevisiae,Aspergillus niger, Fusarium venenatum, Trichoderma reesei,Colletotrichum gloeosporioides, Neurospora crassa andAgaricus bisporus [15, 21, 41]. A recent report even demons-trated DNA transfer to human cells [42]. A. tumefaciens Tiplasmid codes for a second transfer system responsible for Ticonjugative transfer between bacteria. This process operatesthrough a functionally and physically independent tightly-regulated tra-system, containing tra and trb genes, which areequivalent to dtr and mpf genes, respectively [36]. A thirdsystem is featured by avhB encoded proteins, promotingconjugal transfer [43]. Furthermore, a chromosomal tra-likeregion has been recently identified in an Ti-free A.tumefaciens strain, raising the question of the role of such achromosomal region during evolution [44].

Some microbial facultative intracellular pathogens,which withstand bactericidal activities of polymorphonuclearcells, survive within professional phagocytes and multiplyinside the phagosome [45]. They live in intracellularvacuoles until the cell lyses and releases bacteria that startnew rounds of infection [46]. These pathogens possess aT4SS believed to export macromolecules to the vacuolarmembrane or to the cytoplasm of the host cell, which adaptthe vacuolar environment to the bacterial needs, allowingintracellular survival and replication [15, 16, 47]. Brucellosisor Malta fever provokes chronic infections and bacteremia inmammals, including humans, and is caused by Brucella suis,abortus and melitensis [17, 48, 49]. Their genomes alsoencode putative T4SSs consisting of 12 open reading frames(orfs) with homologues of the 11 VirB proteins of A.tumefaciens plamid Ti, with a similar genetic organisation,and a twelfth one, putatively coding for a lipoprotein [17, 48,50-52]. This 12-gene operon is specifically induced byphagosome acidification in cells after phagocytosis. A recentreport on the genome sequence of typhus-causing Rickettsiaprowazekii [53] has revealed seven homologues of VirBproteins of A. tumefaciens, but not of the T-DNA-boundVirD2 and VirE2 proteins. Legionella pneumophila, thecause of Legionnaire’s disease, a form of lobar pneumonia[54, 55], contains four icm/dot genes with significantsequence similarity to A. tumefaciens Vir proteins and thoseof various conjugal transfer systems [56]. This system isessential for apoptosis induction in human macrophages [57]and for secretion of protein DotA [58]. As reported for theVirB transporter, the icm/dot system transfers E. coliRSF1010 plasmid between bacteria, probably as anucleoprotein complex [33, 59, 60]. Like A. tumefaciens, L.pneumophila encodes a second type-IV translocon, notinvolved in pathogenesis, containing 11 lvh genes arrangedsimilarly to the icm/dot genes. This apparatus is dispensablefor cellular growth and it can replace some components ofthe icm/dot system to enable RSF1010 conjugation [61].Most recently, Ehrlichia phagocytophila and chaffeensis, theethiological agents of granulocytic and monocyticehrlichioses and causing febrile systemic illness in humans,and Ehrlichia canis, responsible for canine ehrlichioses,have also been shown to harbour a T4SS [47, 62].

Further bacterial pathogens using T4SSs transfer toxicproteins. These systems may employ the Sec system forprotein translocation across the inner membrane and prior tosecretion [9, 63, 64]. In Helicobacter pylori , the microor-ganism responsible for gastritis, peptic ulcer and gastriccancer, a pathogenicity island (cagPAI), acquired byhorizontal transfer, is the major determinant of virulence.This island codes for 31 Cag proteins, at least six of whichmay contribute to a type-IV system, which translocates in anexample for direct trans-kingdom protein delivery the 150-kDa CagA antigen into gastric epithelial cells, where it istyrosine phosphorylated [65, 66]. There, the protein mayinterfere with the normal host cell signalling inducingpedestal formation, synthesis of proinflammatory cytokinesand chemokines, like interleukin 8, cell spreading andmotiliy, expression of the protooncogenes c fos and c jun,among other events [16, 67, 68], in a phenotype referred toas “hummingbird”. It occasionally enters epithelial cells andmanipulates the host immune system for immune evasion


[69]. Like A. tumefaciens, this pathogen possesses a conjuga-tion-like mechanism of DNA transfer [69-72]. In the closelyrelated Helicobacter hepaticus, three basic components of aT4SS have been found on a genomic island, suggesting thepresence of an orthologous system [73]. Yersinia enterocolitica29930, producing the phage-tail resembling bacteriocinenterolicin harbours a T4SS encoded on a plasmid. It is aconjugative transfer system closely related to the Mpf systemof plasmid R6K. However, an orthologue to TaxB of thelatter system is missing [74]. Finally, Bordetella pertussis,the causative agent of whooping cough in humans, has co-opted a set of conjugal transfer system genes, ptl (frompertussis toxin liberation) [75]. Ptl proteins are highly similarto VirB proteins and direct the export of the A-B-typeendotoxic Pertussis toxin, composed of six subunits, acrossthe bacterial envelope and into the medium [21].

Additional candidate type-IV translocons, or at leastsome constituents, can be found in pathogenic and non-pathogenic organisms and in reported bacterial genomesequences (Table 1).

TRANSFER FACTORS IN CONJUGATIVE TYPE-IV

SECRETION

The described type-IV transport systems require, in addi-tion to the apparatus directly responsible for macromoleculartrafficking, an aiding transfer factor or coupling protein [76,77], when performing conjugation-like nucleoproteintransfer. Such a protein is encountered in all self-transmissible plasmids of Gram-negative bacteria and inmany of Gram-positive bacteria [26]. There is strongevidence that these proteins are also required for type-IVmediated protein export, as reported for A. tumefaciensVirE2 and VirF protein export (in addition to the T-strand /VirD2 complex) and H. pylori [14, 16, 21, 35, 78]. On theother hand, type-IV systems responsible for only proteinexport, like the Bordetella Ptl system, may lack T4CPs.

This class of coupling proteins are integral inner-membrane proteins, with an N-terminal transmembrane partand a C-terminal cytoplasmic moiety, that may assemble ascomponents of the cognate type-IV transport machinery ormay be required to localise near the transport apparatus [79-87]. They bear Walker A (motif I) and B (motif II or DExxbox) NTP-binding sequences [88] and may use the energy of

Table 1. Further Organisms Displaying (Potential) Type-IV Secretion Systems

Organism Type

Organism Name Associated Pathology/Activity References

Mesorhizobium loti bacterium; N2-fixation [153]

Rhizobium etli bacterium; N2-fixation [154]

Sinorhizobium meliloti bacterium; N2-fixation [14,15,17]

Shigella flexneri plasmid ColIb P9 bacterium; diarrhoea / dysentery [155]

Bartonella henselae and tribocorum bacterium; cat scratch disease, bacteremia [156-160]

Campylobacter jejuni bacterium; bacterial diarrhoea [161,162]

Toxoplasma gondii protozoon; toxoplasmosis (encephalitis) [54]

Leishmania donovani protozoon; leishmaniasis [54]

Mycobacterium tuberculosis bacterium; tuberculosis [54]

Bordetella bronchiseptica bacterium; bronchitis [14,15,17]

Enterobacter aerogenes bacterium; nosocomial infections [11]

Actinobacillus actinomycetemcomitans bacterium; periodontitis [14,15,17,163,164]

Escherichia coli bacterium; diarrhea [11]

Caulobacter crescentus bacterium; non-pathogenic [14,15,17]

Coxiella burnetii bacterium; acute febrile disease, endocarditis, pneumonitis [165,166]

Thiobacillus ferroxidans bacterium; iron oxidation [14,15,17]

Wolbachia intracellular symbiont of arthropods; sexual alterations in host [167,168]

Ralstonia eutrophantus bacterium; heavy-metal resistance [11,169]

Salmonella typhi, typhimurium and enteriditis bacterium; typhus, salmonellosis [11,170]

Xylella fastidiosa bacterium; citrus variegated chlorosis [11]


NTP hydrolysis to connect transmissible (nucleo)proteincomplexes with the cognate translocation system [24, 67, 76,82, 84, 89-92]. Mutational studies have revealed that inactiveT4CP mutants affecting NTP binding and/or hydrolysis leadto conjugation-deficient phenotypes [24, 79, 82, 83]. Conju-gation inhibition may also occur when T4CPs associate withother proteins, as shown for RP4 TraG when it interacts withFopA of pKM101 and PifC of plasmid F [89]. Studies onchimeric systems comprising the transport apparatus of onespecies and the T4CP of another reveal that specificityresides on the latter [93, 94]. In some cases, as found forTraD from F, their C-terminal parts affect efficiency andspecificity [95].

TrwB from E. coli plasmid R388, TraD and TraGproteins from various Gram-negative plasmids, VirD4 fromA. tumefaciens Ti and related proteins are T4CPs associatedwith nucleoprotein mobilisation (Table 2). They can beunambiguously aligned using the hidden Markov modelmethod SAM T99 [96] and they are divisible into subfami-lies. The first, which includes TrwB and TraD, contains sixmembers of conjugative plasmids. A second subfamilygroups a broad set of conjugative plasmids (like TraG ofRP4), and the VirD4 protein of Ti plasmids involved in T-DNA transfer to plant cells (Table 2). Proteins within asubfamily are >30% identical in amino acid sequence anddisplay the same domain organisation (Fig. (1b)). In parti-cular, TrwB displays 21-23 % sequence identity to VirD4 ofA. tumefaciens Ti [19] and TraG of RP4 and R751, and 28-30% to TraD of F and R100 [84, 85]. The orthologue fromplasmid R3888 binds both ss and double stranded (ds) DNAwith approximately the same affinity as supercoiled dsDNA.This DNA binding ability is unspecific and independent ofNTP binding. TraD from plasmid F, TraG from RP4 andHP0524 from H. pylori have also been reported to bind ssand dsDNA non-specifically [81, 97]. None of theseorthologues displayed detectable NTP-hydrolysing activityin vitro under the conditions assayed. TrwB is inserted onthe cytoplasmic side of the inner membrane, as describedalso for TraD and VirD4 [79-81, 87]. The latter has a uniquecellular location to the donor cell pole and both itsperiplasmic and the cytosolic parts are required for polarlocalisation, indicating where the associated type-IVsecretion system may also be situated [79]. Some of theseproteins can be functionally exchanged for each other. Thus,TraG can substitute TrwB in the mobilisation of plasmidsColE1 and RSF1010 by R388, but not in self-transfer ofR388 [93]. Evidence has also been found for the interactionpartners of T4CPs. TraG from RP4 interacts with therelaxosome relaxase [92, 97]. In plasmid F, TraD contacts anaccessory nicking protein, TraM [98], and in plasmid R27,TraG interacts with the T4SS protein TrhB [99].

T4CPS AND SPOIIIE / FTSK PROTEINS

T4CPs and members of the SpoIIIE/FtsK protein familyshare functional and structural features (Fig. (1b); [82, 100-102]). SpoIIIE is an essential sporulation membrane-boundprotein in Bacillus subtilis , whose cytoplasmic part interactswith the chromosomal DNA to achieve DNA translocationfrom the mother cell through an annulus to the prespore. It is

also responsible for the polarity of DNA transfer [103].Several orthologues of SpoIIIE have been reported inBacillus halodurans, Sporosarcina ureae and Streptomycescoelicolor. SpoIIIE displays significant sequence similarityto Tra proteins of several Gram-positive conjugative plas-mids of Streptomyces [101]. The annulus may be lined up bya number of SpoIIIE proteins to render a continuous ring,through which the prespore chromosome may translocate[104]. The C-terminal cytoplasmic transfer domain ofSpoIIIE shows ATPase activity and the protein can trackalong DNA in the presence of ATP [100, 102]. The proteinbehaves as a DNA pump that actively moves one of thereplicated pair of chromosomes into the prespore [100]. Onthe other hand, E. coli FtsK is involved in the resolution ofchromosomal dimers during bacterial cell division [105]. Itprecisely synchronises the separation of the two daughterchromosomes by XerCD recombinase through site-specificrecombination [106]. FtsK may also pump newly replicatedchromosomes away from the septal space during septation[105]. Orthologues of FtsK, which is essential, are probablypresent in all bacteria (to date reported for Campylobacterjejuni, Coxiella burnetii, Haemophilus influenzae, Proteusmirabilis, H. pylori, Azospirillum brasilense, Chlamydiatrachomatis, muridarum and pneumoniae, S. coelicolor,Pseudomonas aeruginosa and syringae, Neisseria meningi-tides, R. prowazekii, Deinococcus radiodurans, Ureaplasmaparvum and Vibrio cholerae), but also in eukarya (Caenor-rhabditis elegans). The SpoIIIE/FtsK analogues found todate are YPTP from B. subtilis, Tra/KilA from Streptomyceslividans plasmid IJ01 and Trasa/Integrase fusion protein,among others. T4CPs have the same molecular organisationas the C-terminal domain of SpoIIIE/FtsK-like proteins (Fig.(1b)) and share extensive sequence similarities, especiallyaround the NTP-binding Walker motifs. This suggests afunctional link, as an NTP-dependent pump that transfersnucleoprotein complexes or proteins via type-IV translocons.

THE FOLD AND QUATERNARY STRUCTURE OF AT4CP

The 33-kb plasmid R388 [107] contains genes that codefor sulphonamide and trimethoprim resistance [108, 109]. Itsmpf region encodes a functional type IV secretion system,featuring 11 trw genes (trwD to trwN). R388 displays a shortdtr region made up by oriT, trwA, trwB, and trwC [85, 110].TrwC acts as a relaxase and helicase. It is responsible forboth nick-cleavage at oriT and T-strand unwinding, and itsthree-dimensional structure in complex with oriT DNA hasbeen solved [111-113]. TrwA is a small-sized tetramericprotein, which binds two sites at oriT and enhances TrwCrelaxase activity, while repressing transcription of thetrwABC operon [114]. These two proteins constitute,together with oriT DNA and the host-encoded integrationhost factor (IHF), the relaxosome of plasmid R388 [85]. IHFhas a modulator role in conjugation [115], as it inhibitsTrwC nic cleavage, probably by affecting the topology of theTrwC DNA target site [116].

The third R388 dtr protein is TrwB, the T4CP of thissecretory system [82, 85]. The structure of the cytoplasmicpart of TrwB can be divided into an all-α domain and a

Table 2. Some Members of the Type-IV Coupling Protein (T4CP) Family


NameOrganism

(p=plasmid)

Data Bank access code

(GB, GenBank; SP, SwissProt/

TrEMBL; PIR, Prot.Inf.Res.)

Reference

TrwB

TraD

TraJ

Escherichia coli R388

Xanthomonas axonopodis pXAC64

E. coli R100

E. coli F

Sphyngomonas aromaticivorans pNL1

pKM101

pCU1

SP Q04230

SP Q8PRK2

SP P22708

SP O87742, P09130

SP 085919

SP O06958

SP Q9X4G1

[82,85]

[171]

[172]

[173]

[174]

[175]

[176]

VirD4

LvhD4

TraG

Agrobacterium tumefaciens/rhizogenes Ti

A. tumefaciens/rhizogenes Ri

Wolbachia sp. wKueYO and wTai

Rickettsia prowazekii

Rickettsia conorii

Legionella pneumophila

E.coli RP4

E. coli R751

E. coli R64

SP P09817, P18594, Q8VT85, Q8VLK3

SP P13464, Q9F585

SP Q9KW36, Q9KW43

SP Q9ZDNA

SP Q92IM9

SP Q9RLR2

SP Q00185

SP Q00184

GB AB027308

[19]

[177-179]

[168]

[53]

[180]

[61]

[31]

[181]

[182]

TraG

TraK/VirD4-like

TraN/VirD4-like

TrsK

TaxB

Cag524/HP0524/Cag-β

IcmO/DotL

MobC

MobB

Hypot. Protein SCE2.07

TrbB

Orf6

VirD4-like protein

VagA

VirD4-like sex factor protein

Helicobacter pylori

Xylella fastidiosa pXF51

Actinobacillus actinomycetemcomitans pVT74 (MAGB12)5

E. coli R721

Rhizobium sp. PNGR234

Rhizobium loti pMLb

A. tumefaciens/Rhizobium sp. Ti

A. tumefaciens/Rhizobium sp. Ri

Ralstonia solanacearum

Pseudomonas sp. PADP-1

Streptococcus pneumoniae Tn5252

Haemophilus aegyptus pF3031

Clostridium acetobutylicum

alfalfa rhizosphere microbial community pSB102

Stapylococcus aureus pGO1/pSV41

Lactococcus lactis pMRC01

Enterococcus faecalis Tn1549 in pIP834

E. coli R6K

H. pylori

L. pneumophila

Bacteroides fragilis

Enterobacter cloacae CloDF13

Streptomyces coelicolor

ColIB-P9

Pediococcus pentosaceus pMD136

X. axonopodis

X.campestris

E. coli pIPO2T

Anaplasma phagocytophylum

Ehrlichia chaffeensis

R. loti

X. campestris

L. lactis 712

SP O25252

SP Q9PHI9

SP Q9F254

SP Q9F531

SP P55421

SP Q98NT6, Q981R7, GB CAD31437

SP Q44346

GB NP_066690

SP Q8XW89

SP Q937B8

GB AAG38037

SP Q8VRC7

SP Q97HP1

SP Q9IUR9

SP Q07713

SP O87219

GB AAF72343

SP O32582

SP O25260, Q9ZLV3, Q9JMY0, GB

AAF80195

PIR T18329, SP O54524

SP Q9ZF54

SP P08098

PIR T36167

GB NP_052502

SP Q9WW76

GB AAM37472

GB AAM41759, AAM42732

SP Q91UW5

SP Q8RPL3

SP Q8RPL9

GB CAD31465

SP Q93TC4

–

[183]

[184]

[185]

direct submission

[186]

[187]

[188,189]

direct submission

[190]

direct submission

[191]

direct submission

[192]

[193]

[194]

[195]

[196]

[197]

[16,183]

[60]

[198]

[199]

direct submission

direct submission

[200]

[171]

[171]

[201]

[47]

[47]

[153]

direct submission

[202]

(Table 2) contd….


NameOrganism

(p=plasmid)

Data Bank access code

(GB, GenBank; SP, SwissProt/

TrEMBL; PIR, Prot.Inf.Res.)

Reference

TrsK-like protein

TrsK-like protein

TraG-like protein

Putative transposase

?

?

?

Rhodococcus equi p33701

Bacillus anthracis pXO2

Sulfolobus sp. pINE1

Sulfolobus sp. pNOB8

Klebsiella pneumophila pR55

Salmonella typhimurium

L. pneumophila

Mycobacterium tuberculosis

GB NP_066787

GB NP_053171

GB AF233440

SP O93672

SP Q8VW38

gnl|WUGSC 99287|stmlt2-.Contig1522

gnl|CUCGC 446|lpneumo MF-

473.111897(different from LvhD4)

gnl|WIGSC 573|kpneumo B

KPN.Contig1729

[203]

[204]

[205]

[206]

[207]

Searches performed against SwissProt/TrEMBL at www.expasy.ch and against GenBank and PIR at www.ncbi.nlm.nih.gov:80/entrez. Similar domains were looked for withPRODOM2000.1, www.toulouse.inra.fr/prodom.html. “?” indicate searches performed against incomplete bacterial genomes at www.ncbi.nlm.nih.gov/Microb_blast/unfinishedgenome.html. Representatives belonging to the same subfamily are framed (see Fig. (1b)).

Fig. (1). (a) Model of the bacterial conjugative process (see also [77]). � A donor cell (blue contour) possessing a plasmid contacts arecipient cell (light green contour) lacking this genetic element. Intercellular contact is mediated by the pilus and type-IV secretory proteins ofthe plasmid mpf region. A mating signal, putatively generated by this contact between the donor pilus and the receptor, apparently leads to aspecific interaction between the relaxosome and the T4CP at the inner face of the conjugative pore [32, 92, 97]. The supercoiled dsDNAplasmid interacts via its oriT region with proteins of the plasmid dtr gene locus creating the relaxosome. � The pilus putatively retracts topermit intimate contact between bacterial cell walls. The connecting pore or tunnel is formed as a result of mating-pair stabilisation. � TheDNA-strand transferase / helicase or relaxase enzyme of the relaxosome introduces a strand-specific (T-strand) nick at oriT. The relaxosomeinteracts with the inner-membrane-anchored T4CP. �� Transfer of the T-strand through the tunnel is putatively initiated, and both generatedssDNAs replicate following the rolling circle mechanism (dotted lines). � Once the transfer is finished, the free ends of the plasmids arejoined and the pore closes, the acquired surface exclusion disrupts aggregation, and mating bacteria separate. Finally, the identical plasmidssupercoil. Both resulting cells are now capable of a subsequent conjugation event. (b) Schematic alignment of some representatives of theT4CP family with their length in amino acids. The first subfamily is exemplified by TrwB (plasmid class IncW) and TraD (IncF). A secondsubfamily encompasses TraG (IncP) and VirD4 proteins [77]. Finally, a distinct group of proteins is involved in bacterial cell division(exemplified by FtsK of E. coli) and in sporulation of Gram-positive bacteria (related to SpoIIIE of B. subtilis). TrwB and VirD4 subfamilies[93] are more closely related than they are to FtsK/SpoIIIE proteins [101, 208]. Periplasmic transmembrane segments (beige rods) arerepresented, separating short periplasmic regions (left) from large cytosolic ones (right). These are mainly made up by the NBDs (darkbrown) with the A and B walker motifs forming the nucleotide-binding site indicated. The all-α domains (light brown) present in the TrwBand VirD4 subfamilies do not share significant sequence similarity, although they are both inserted into their respective NBDs. (c) Modeldepicting a possible arrangement of the 11 mpf encoded Trw proteins (TrwD-N) making up the type-IV translocon engaged in T-stranddelivery from the donor cell to the recipient cell during conjugation of R388. The arrangement is based on the proposed locations for thehomologous VirB proteins [2, 11, 15, 16, 19, 21, 40, 71, 85, 86, 209].


nucleotide-binding domain (NBD) attached to the innermembrane, while the first seventy N-terminal residuesfeature two transmembrane helices flanking a smallperiplasmic region in between ([2, 117]; Figs. (1b) and (2a)).The NBD features an α/β P-loop-containing NTP hydrolasecore. On the membrane-proximal edge of this central β-sheet, a small three-stranded antiparallel sheet is placed,almost perpendicularly. Its constituting strands are flexible,owing to the absence of interactions with the transmembranedomain preceding strand 1 (excised in the experimentalstructure; [117, 118]), which has been tentatively modelled(Fig. (2a)). At the tip of the NBD, at the membrane-distalpart, the smaller, entirely helical all-α domain is positioned.

Six identical protomers shape a spherical particle,flattened at both poles along its vertical axis, of 90 Å inheight and 110 Å in diameter (Fig. (2c)). This is consistentwith biochemical data for the native protein migrating as ahexamer in gel filtration column chromatography (ourunpublished results). A central channel runs from the cyto-solic surface (made up by the upper all-α domains) to themembrane-proximal pole (formed by the NBDs), ending atthe transmembrane pore shaped by the transmembranesegments (Figs. (2a, b)). This channel is limited to adiameter of ∼7-8 Å at its cytoplasmic side. This is the closestpoint of the channel that, at its membrane end, has anopening of ∼22 Å.

STRUCTURAL SIMILARITIES

The family of AAA proteins, described for eukaryotes,prokaryotes and archaebacteria, includes ATPases associatedwith a variety of cellular activities that display an NBD witha fold similar to TrwB NBD. It is an essential family of spe-cialised, chaperone-like enzymes that participate in membranefusion and trafficking, organelle biogenesis, proteolysis andprotein folding [119, 120]. It includes the δ’ subunit of theclamp loader complex of Escherichia coli DNA polymeraseIII (Protein Data Bank access code (PDB) 1a5t; [121]), N-ethylmaleimide-sensitive fusion protein domain 2, acytosolic ATPase required for intracellular vesicle fusionreactions (PDB 1d2n; [122]), AAA ATPase p97, involved inhomotypic membrane fusion (PDB 1e32; [123]), and ATP-dependent protease HsIU-HsIV (PDB 1e94; [124]).

RNA and DNA helicases are molecular motors involvedin DNA metabolism using the energy from NTP hydrolysisfor nucleic-acid unwinding or strand separation andtranslocation [125]. Despite a substantial body of bioche-mical and structural data, it is only in the past couple ofyears that this information has unveiled parts of the workingmechanism [126]. The helicases that unwind dsDNA at thereplication fork are ring-shaped hexamers that catalyse thedisplacement of the complementary strand [127, 128]. Otherproteins known as RNA helicases use the chemical energy ofNTP hydrolysis to remodel the interactions of RNA andproteins [129]. The recently reported crystal structures oftwo toroidal DNA ring helicases, those of the T7 phage gene4 protein [130] and RepA [131], suggest a sequential NTP-hydrolytic mechanism. E. coli RecA, whose hexamericarchitecture was first described by electron microscopystudies [132], is the structural prototype for these enzymes[132-134]. As a key ATP-dependent exchange factor, it is

essential to genetic recombination and repair. Extensivestructural studies, including several complexes, have beencarried out with monomeric 3’-5’ PcrA DNA helicase, aprotein that binds both dsDNA, prior to strand separation,and ssDNA, after separation, to avoid annealing [126, 135,136]. TrwB bears structural similarity within its NBD withthe equivalent part of RecA (PDB 2reb) and other RecA-likecore encompassing enzymes, such as the replicativehelicase/primase of bacteriophage T7 (PDB 1cr0; [127]),besides to T7 gene 4 ring helicase (PDB 1e0j; [130]) andPcrA DNA helicase (PDB 2pjr and 3pjr; [136]). Finally, therecently solved six-clawed grapple-shaped structure ofhomohexameric H. pylori Cag525/HP0525 traffic ATPase,an inner-membrane associated part of the bacterial type-IVsecretion system involved in pathogenic protein CagA export(see above) and the TrwD orthologue in the R388 system[137], reveals also structural similarity in the NBD core[138].

Several representatives of the helicase/ATPase-likeproteins display, in addition to a NBD, an all-α domain, asshown for RecA, AAA ATPases, PcrA DNA helicase, andthe α and β subunits of F1-ATPase, among others, althougharranged in distinct ways with respect to their NBDs.However, the TrwB all-α domain bears significant structuralsimilarity only to N-terminal domain 1 of the site-specificrecombinase, XerD, of the λ integrase family (PDB 1a0p;[139]). This domain is positioned over the DNA bindingregion blocking the access to DNA. A large conformationalrearrangement is therefore required for target DNA binding.This rearrangement may also occur in TrwB, which wouldprovide a putative working mechanism (see below). XerDsite-specific recombination has been associated with FtsK[105], a protein that shares functional and structural featureswith TrwB and other T4CPs (see above). XerD works inconjugation with FtsK to resolve the knots formed afterreplication of the bacterial chromosome. If TrwB AAD wasthe functional counterpart of XerD it could be involved inDNA binding or some form of processing. A central pore isalso shaped by α helices from the constituting subunits insix-clawed grapple-like Cag525. These subunits do notcomprise a distinct subdomain, but they are localised on theconvex side of the central β-sheet of the NBD [138].

The TrwB all-α domain displays further topologicalsimilarity within a ~40-residue segment encompassing threeα-helices with the recently reported solution structure of the56-residue DNA-binding domain of TraM, a component ofthe relaxosome encoded by the dtr region of plasmid R1[140]. This protein enhances relaxase activity and it iscapable of binding DNA [141]. TraM specifically interactswith the TrwB orthologue in IncF plasmids, TraD,suggesting that TraM links the relaxosome with the DNAtransfer apparatus [98]. Plasmid R388 lacks such a TraMorthologue. Therefore, an appealing hypothesis is that TrwBhas imbedded in its structure a DNA-binding domain similarto that of TraM, that may directly recruit the relaxosome.

Nevertheless, on examining the hexameric toroidalquaternary structures of the protein families mentioned (Figs.(2c-f)), helicases, AAA ATPases and Cag525 appear moreflat-topped than TrwB, with much less interaction surfacebetween the constituting protomers. This is needed in


Fig 2. (a) Ribbon diagram of a TrwB protomer, with its transmembrane domain tentatively modeled (gray). The constitutingdomains and termini are further depicted. (b) Connolly electrostatic surface (blue, positive patches; red, electronegative parts;modelled transmembrane part in yellow) showing only four of the six protomers to visualise the deep central channel traversingthe particle. (c) Ribbon plot of the hexameric ring-like structure of (transmembrane-depleted) TrwB [117], (d) α3β3

heterohexameric F 1-ATPase [143], (e) H. pylori Cag525/HP0525 traffic ATPase [138], and (f) T7 gene 4 DNA ring helicase[130]. The monomers are shown alternatively in green and yellow. For each particle, axial (left) and lateral (right) views arepresented.


helicases to allow interchanging aggregation stages andhelicoidal protein-filament formation [126, 127, 142]. Thehexameric structure of TrwB, with almost 50% of the surfaceof a monomer shaping the interface to vicinal protomersalmost rules out other oligomerisation states. Most strikingstructural similarity of the TrwB oligomer is found to both αand β subunits of F1-ATPase, part of the membrane-associated F0F1-ATPase complex responsible for energyconversion through axial rotation movement in mitochon-dria, chloroplasts, and bacteria (PDB 1bmf; [143]), sugges-ting also a link on the functional level (see below). Theoverall hexamer dimensions and the almost spherical shapeare much more reminiscent of the F1-ATPase α3β3 hetero-hexamer. Like TrwB, this protein is a membrane protein.

WORKING HYPOTHESIS FOR CONJUGATIVET4CPS

The mechanism of action of type-IV secretion has notbeen experimentally established yet for all its steps. Hereby,we discuss alternative hypothetical functions for TrwB andother T4CPs, that may act as a motor pushing the T-DNA tothe transport apparatus, as simple bridging proteins betweentwo macromolecular assemblies, the relaxosome and thetype-IV transport machinery, or as true DNA pumps thatthread the T-strand through the pore connecting the donorand the recipient cells. This latter function could be part of a“shot and pump” model, following which T4CPs wouldcatalyse active pumping of T-DNA to the recipient once thetype-IV secretion system has shuttled a pilot protein, therelaxase and helicase (TrwC in the R388 system, see above),with the covalently bound T-DNA through the pilusappendage to the acceptor cell [77]. In the first and thirdT4CP functional hypotheses, ATP hydrolysis, probablytriggered by interactions with other cellular components, likethe relaxosome itself, or with membrane lipids, should benecessary. Furthermore, this hydrolytic activity might beassociated with an axial rotating motion to translocate DNA.

The first model portrays TrwB as a rotary machine. TheTrwB hexamer displays an exterior surface belt with highlypositive charges that may explain the capability of theprotein to bind ss and dsDNA non-specifically [82]. Accord-ingly, ssDNA, after being processed by the nicking enzymeand helicase TrwC, may wrap around a TrwB hexamer (Fig.(3a)). The hexamer could behave as a membrane-anchoredmotor spinning around its vertical axis (following the F0F1-ATPase analogy). Relaxosome T-DNA would be transloca-ted by TrwB to the mating apparatus and there cross the poreto the recipient cell. Electron microscopy images ofelongated aggregates of TrwB molecules and DNA [82] maysupport this hypothesis, but there is no further evidence forthis wrapping model. Mutation studies concerning basicresidues of the belt show no functional implications.Therefore, this model is probably the less likely one.

The second hypothesis implies that the coupling proteinis not a pump or motor at all. It would behave just as a two-sided recognition protein recruiting the relaxosome bycontacting its components on one side and the inner-membrane part of the type-IV transport machine on anotherside (Fig. (3b)). This model does not require an ATPaseactivity, although a nucleotide-binding-induced molecular

switch is reasonable. Furthermore, it would be difficult toexplain the presence of a nucleotide-binding site otherwise.No direct ATPase activity has been observed forTrwB∆N70, but this is not a concluding result, sincecleavage of the transmembrane part may have hindered theenzyme activity. The completely transfer-deficient K136Tmutant of TrwB binds ATP normally, so deficiency must besomehow related with hydrolysis. Moreover, some othercomponents - relaxosome, DNA, etc. - may be required totrigger TrwB functionality.

The structure of H. pylori Cag525 protein [138], amember of the VirB11/PulE family of ATPases [144]involved in the type-IV secretion system of the latterpathogen, reveals a six-clawed grapple shape mounted on ahexameric ring and it is proposed to be membrane-asso-ciated. It has been shown to function as dynamic hexamericassemblies with nucleotide-dependent conformationalchanges regulating substrate export or T4SS assembly [145].The membrane-distal surface of the hexamer is formed by ahelical bundle at the C-terminus of the RecA-like α/βdomain. The six bundles restrict the size of the central poreto ∼10 Å (Fig. (2e)). An orthologue of this protein is alsopresent in the secretory organelle of plasmid R388 in theform of TrwD [146]. Except for the RecA-like α/β core andthe hexameric nature of the protein, the TrwB and Cag525structures strongly differ. As the latter ATPase may parti-cipate in the trafficking of 150-kDa CagA protein to gastricor duodenal epithelial cells during pathogenesis of H. pylori,the authors propose a mechanism of ATP-dependent opening/closure of the pore following domain rearrangement andsubsequent protein export through this pore [138]. If theequivalent TrwD plays such a role in the conjugation systemtransporting DNA or a protein/DNA complex instead of theCagA toxin, TrwB would probably not have the samefunction. However, TrwB has a real transmembrane domain,whereas the evidence for TrwD or Cag525 protein crossingthe membrane is more circumstantial.

Finally, ssDNA may pass through the central hexamerchannel, may be injected through the inner membrane intothe periplasmic space and there to the translocon (Fig. (3c)).By the reasons mentioned before, this is the preferredalternative. Thus, conjugal T4CPs would be behaving asNTP-dependent DNA pumps that transfer the T-strandthrough a type-IV system during conjugation, in a fashionsimilar to SpoIIIE/FtsK-like proteins or as has been postu-lated for RuvB, which is capable of dsDNA translocation[100, 101, 147]. The channel is basically of electronegativenature, but it possesses belts of positively charged (arginineand lysine) amino acids (Fig. (2b)). Taking into account thatthe passing DNA would slip smoothly through the channel,an extended electropositive surface would not be adequate.This has been seen in a channel crossing the structure of areovirus, which is traversed by a RNA molecule [148], andfor the bacteriophage Φ29 connector particle [149, 150]. Theside of the TrwB channel facing the inner-membrane has anopening of ∼ 22 Å, compatible with the values of 25-30 Åfor RecA-like hexameric helicases, through which ssDNAmay pass [151]. Functional T7 gene 4 ring helicase has aneven narrower central hole, of ~15 Å [130]. The mainobjection to this hypothesis is the narrow entrance to thechannel (∼7-8 Å). However, constriction here may impose a


level of regulation that prevents continuous leakage of thecell contents. On the other hand, the bacterial cell mayprevent permeation of the membrane for ions and smallmolecules through this narrow though open channel. Thus,the channel may be even further constricted at thetransmembrane part, remaining impermeable until a certainstimulus triggers the opening, e.g. interaction with theforming relaxosome. This may occur by domainrearrangement, as has been proposed for the structurallysimilar domain 1 of XerD (see above). Accordingly, a slightmotion around the hinge region between the all-α domainand NBD may increase the channel diameter to permitssDNA threading (Fig. (3c)).

CONCLUSION

With the present data, it is still difficult to define theprecise mechanism of DNA transport during conjugation,but, for the first time, crucial structural data on three of theconstituting parts of type-IV secretion systems and theiraccessory proteins are available (TrwB, Cag525 and TraM).The first two have been found to be toroidal hexamers. Suchmultimeric ring-shaped structures are associated with distinctactivities like proteolysis, folding assistance, migration alongnucleic acids, cell-cycle regulation and proton pumping,among others [152]. A toroidal protein oligomerisation ischaracterised by the presence of a channel or pore traversing

the particle and thus connecting two environments that maydiffer or forming a cavity that harbours the catalytic site(s).When the oligomerisation involves a single or similarprotomers, the particle contains several (potential) activesites. Formation of a toroid via oligomerisation has theadvantage that construction of a circular structure bymultiplication and assembly of equal subunits is simpler andrequires less genetic information than the design of one largeprotein with a central cavity. Moreover, it generates multipleidentical binding sites that may allow sequential binding andrelease of substrates or interacting molecules withoutdissociation from the ring, as suggested for ring helicaseswhile translocating along nucleic acids. Another advantage isthe formation of a topological link with the transportmacromolecule, avoiding dissociation and conferringprocessivity, and thus achieving the speed necessary formanipulation of large macromolecules within the short timeframe required for translocation or secretion.

ACKNOWLEDGEMENTS

This study was supported by grants BIO2000-1659,BIO2002-00063, BIO2002-03964, and BIO2003-00132 (toM.C and F.X.G.R) and by BMC2002-00379 (to F.C.) fromthe Ministerio de Ciencia y Tecnología, Spain, and by grant2001SGR346 and the Centre de Referència en Biotecnologia,both from the Generalitat de Catalunya.

Fig. (3). Working model for TrwB and other T4CPs. The dtr region of E. coli plasmid R388 is just made up of oriT, trwA, trwB, and trwC[85, 110]. TrwC behaves as a relaxase and a helicase and it is responsible for both nick cleavage at oriT and T-strand unwinding beforetransfer [112, 113]. TrwA is a small, tetrameric protein with a dual role in conjugation [114]. It binds to two sites at oriT and enhances TrwCrelaxase activity while repressing transcription of the trwABC operon. Both proteins, TrwA and TrwC, build up the relaxosome in R388,together with oriT DNA and the host-encoded integration host factor [2, 114]. After putative nicking at oriT and DNA unwinding, therelaxosome contacts the coupling protein. (a) Hypothesis suggesting that the T-strand DNA wraps around the particle. Vertical spinningenergised by ATP hydrolysis may promote transfer to the transport pore. (b) Hypothetical model of the relaxosome recruited by TrwB,which in turn is attached to the type-IV translocon, allowing T-DNA transfer. (c) Hypothesis proposing that the T-strand threads through thecentral channel upon rearrangement of the all-α domains to enlarge the cytoplasmic entrance. In this case, ATP-mediated spinning orsequential rearrangements in the channel may allow pumping of the T-strand into the periplasm.


REFERENCES

References 210-212 are related articles recently published inCurrent Pharmaceutical Design.

[1] Nagai H, Roy CR. Show me the substrates: modulation of host cellfunction by type IV secretion systems. Cell Microbiol 2003; 5:373-83.

[2] Gomis-Rüth FX, de la Cruz F, Coll M. Structure and role ofcoupling proteins in conjugal DNA transfer. Res Microbiol 2002;153: 199-204.

[3] Plano GV, Day JB, Ferracci F. Type III export: new uses for an oldpathway. Mol Microbiol 2001; 40: 284-93.

[4] Linton KJ, Higgins CF. The Escherichia coli ATP-binding casette(ABC) proteins. Mol Microbiol 1998; 28: 5-13.

[5] Jacob-Dubuisson F, Locht C, Antoine R. Two-partner secretion inGram-negative bacteria: a thrifty, specific pathway for largevirulence proteins. Mol Microbiol 2001; 40: 306-13.

[6] Sandqvist M. Biology of type II secretion. Mol Microbiol 2001; 40:271-83.

[7] Salmond GPC. Secretion of Extracellular Virulence Factors byPlant Pathogenic Bacteria. Annu Rev Phytopathol 1994; 32: 181-200.

[8] Krause S, Pansegrau W, Lurz R, de la Cruz F, Lanka E.Enzymology of type IV macromolecule secretion systems: theconjugative transfer regions of plasmids RP4 and R388 and the cagpathogenicity island of Helicobacter pylori encode structurally andfunctionally related nucleoside triphosphate hydrolases. J Bacteriol2000; 182: 2761-70.

[9] Burns DL. Biochemistry of type IV secretion. Curr Op Microbiol1999; 2: 25-9.

[10] Galán JE, Collmer A. Type III secretion machines: bacterialdevices for protein delivery into host cells. Science 1999; 284:1322-8.

[11] Cao TB, Saier Jr MH. Conjugal type IV macromolecular transfersystems of Gram-negative bacteria: organismal distribution,structural constraints and evolutionary conclusions. Microbiology2001; 147: 3201-14.

[12] Saier Jr MH. A functional-phylogenetic classification system fortransmembrane solute transporters. Microbiol Mol Biol Rev 2000;64: 354-411.

[13] Kostelidou K, Thomas CM. DNA recognition by the KorA proteinsof IncP-1 plasmids RK2 and R751. Biochim Biophys Acta 2002;1576: 110-8.

[14] Christie PJ. Type IV secretion: intercellular transfer ofmacromolecules by systems ancestrally related to conjugationmachines. Mol Microbiol 2001; 40: 294-305.

[15] Christie PJ, Vogel JP. Bacterial type IV secretion: conjugationsystems adapted to deliver effector molecules to host cells. Trendsin Microbiol 2000; 8: 354-60.

[16] Covacci A, Telford JL, del Giudice G, Parsonnet J, Rappuoli R.Helicobacter pylori virulence and genetic geography. Science1999; 284: 1328-33.

[17] O'Callaghan D, Cazevieille C, Allardet-Servent A, Boschiroli ML,Bourg G, Foulongne V, et al. A homologue of the Agrobacteriumtumefaciens VirB and Bordetella pertussis Ptl type IV secretionsystems is essential for intracellular survival of Brucella suis . MolMicrobiol 1999; 33: 1210-20.

[18] Anthony KG, Sherburne C, Sherburne R, Frost LS. The role of thepilus in recipient cell recognition during bacterial conjugationmediated by F-like plasmids. Molec Microbiol 1994; 13: 939-53.

[19] Zupan JR, Ward D, Zambryski P. Assembly of the VirB transportcomplex for DNA transfer from Agrobacterium tumefaciens toplant cells. Curr Op Microbiol 1998; 1: 649-55.

[20] Winans SC, Burns DL, Christie PJ. Adaptation of a conjugaltransfer system for the export of pathogenic macromolecules.Trends Microbiol 1996; 4: 64-8.

[21] Christie PJ. Agrobacterium tumefaciens T-complex transportapparatus: a paradigm for a new family of multifunctionaltransporters in eubacteria. J Bacteriol 1997; 179: 3085-94.

[22] Frost LS, Ippen-Ihler K, Skurray RA. Analysis of the sequence andgene products of the transfer region of the F sex factor. MicrobiolRev 1994; 58: 162-210.

[23] Piper KR, Farrand SK. Quorum sensing but not autoinduction of Tiplasmid conjugal transfer requires control by the opine regulon andthe antiactivator TraM. J Bacteriol 2000; 182: 1080-8.

[24] Firth N, Ippen-Ihler K, Skurray RA. In: Neidhart FC, Curtiss III R,Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS,Riley M, Schaechter M, Umbarger HC Eds, Escherichia coli andSalmonella: cellular and molecular biology. Washington, D.C.,American Society for Microbiology 1996; 2377-401.

[25] Buchanan-Wollaston V, Passiatore JE, Cannon F. The mob andoriT mobilization functions of a bacterial plasmid promote itstransfer to plants. Nature 1987; 328: 172-5.

[26] Zechner EL, de la Cruz F, Eisenbrandt R, Grahn AM, KoraimannG, Lanka E, et al. In: Thomas CM Eds, The horizontal gene pool:bacterial plasmids and gene spread. London, Harwood AcademicPublishers 2000; 87-173.

[27] Lanka E, Wilkins BM. DNA processing reactions in bacterialconjugation. Annu Rev Biochem 1995; 64: 141-69.

[28] Heinemann JA. Genetics of gene transfer between species. Trendsin Genetics 1991; 7: 181-5.

[29] Heinemann JA, Sprague GFJ. Bacterial conjugative plasmidsmobilize DNA transfer between bacteria and yeast. Nature 1989;340: 205-9.

[30] Lessl M, Lanka E. Common mechanisms in bacterial conjugationand Ti-mediated T-DNA transfer to plant cells. Cell 1994; 77: 321-4.

[31] Grahn AM, Haase J, Bamford D, Lanka E. Components of the RP4conjugative transfer apparatus from an envelope structure bridginginner and outer membranes of donor cells: implications for relatedmacromolecule transport systems. J Bacteriol 2000; 182: 1564-74.

[32] Lawley TD, Klimke WA, Gubbins MJ, Frost LS. F factorconjugation is a true type IV secretion system. FEMS MicrobiolLett 2003; 224: 1-15.

[33] Beijersbergen A, den Dulk-Ras A, Schilperoort RA, Hooykaas PJJ.Conjugative transfer by the virulence system of Agrobacteriumtumefaciens. Science 1992; 256: 1324-7.

[34] Heinemann JA. Genetic evidence of protein transfer duringbacterial conjugation. Plasmid 1999; 41: 240-7.

[35] Vergunst AC, Schrammeijer B, den Dulk-Ras A, de Vlaam CMT,Regensburg-Tuïnk TJG, Hooykaas PJJ. VirB/D4-dependent proteintranslocation from Agrobacterium into plant cells. Science 2000;290: 979-82.

[36] Li P-L, Hwang I, Miyagi H, True H, Farrand SK. Essentialcomponents of the Ti plasmid trb system, a type IVmacromolecular transporter. J Bacteriol 1999; 181: 5033-41.

[37] de la Cruz F, Lanka E. In: Spaink HP, Kondorosi A, Hooykaas PJJEds, The rhizobiaceae. Dordrecht, Netherland, Kluwer AcademicPublishing 1998; 281-301.

[38] Tzfira T, Rhee Y, MHC, Kunik T, Citovsky V. Nucleic acidtransport in plant-microbe interactions: the molecules that walkthrough the walls. Annu Rev Microbiol 2000; 54: 187-219.

[39] D'Halluin K, Botterman J. In: Spaink HP, Kondorosi A, HooykaasPJJ Eds, The rhizobiaceae: molecular biology of model plant-associated bacteria. Amsterdam, Kluwer Academic Publishers1998; 339-45.

[40] Lai E-M, Kado CI. The T-pilus of Agrobacterium tumefaciens.Trends in Microbiol 2000; 8: 361-9.

[41] de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen A.Agrobacterium tumefaciens-mediated transformation offilamentous fungi. Nat Biotech 1998; 16: 839-42.

[42] Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V.Genetic transformation of HeLa cells by Agrobacterium. Proc NatlAcad Sci USA 2001; 98: 1871-6.

[43] Chen L, Chen Y, Wood DW, Nester EW. A new type IV secretionsystem promotes conjugal transfer in Agrobacterium tumefaciens. JBacteriol 2002; 184: 4838-45.

[44] Leloup L, Lai EM, Kado CI. Identification of a chromosomal tra-like region in Agrobacterium tumefaciens. Mol Genet Genomics2002; 267: 115-23.

[45] Smith LD, Ficht TA. Pathogenesis of Brucella. Crit Rev Microbiol1990; 17: 209-30.

[46] Shuman HA, Horwitz MA. Legionella pneumophila invasion ofmononuclear phagocytes. Curr Top Microbiol Immunol 1996; 209:99-112.

[47] Ohashi N, Zhi N, Lin Q, Rikihisa Y. Characterization andtranscriptional analysis of gene clusters for a type IV secretionmachinery in human granulocytic and monocytic ehrlichiosisagents. Infect Immun 2002; 70: 2128-38.

[48] Sieira R, Comerci DJ, Sánchez DO, Ugalde RA. A homologue ofan operon required for DNA transfer in Agrobacterium is required


in Brucella abortus for virulence and intracellular multiplication. JBacteriol 2000; 182: 4849-55.

[49] Hong PC, Tsolis RM, Ficht TA. Identification of genes required forchronic persistence of Brucella abortus in mice. Infect Immun2000; 68: 4102-7.

[50] Boschiroli ML, Ouahrani-Bettache S, Foulongne V, Michaux-Charachon S, Bourg G, Allardet-Servent A, et al. The Brucella suisvirB operon is induced intracellularly in macrophages. Proc NatlAcad Sci USA 2002; 99: 1544-9.

[51] del Vecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, Los T,et al. The genome sequence of the facultative intracellular pathogenBrucella melitensis. Proc Natl Acad Sci USA 2002; 99: 443-8.

[52] Delrue RM, Martínez-Lorenzo M, Lestrate P, Danese I, Bielarz V,Mertens P, et al. Identification of Brucella spp. genes involved inintracellular trafficking. Cell Microbiol 2001; 3: 487-97.

[53] Andersson SGE, Zomorodipour A, Andersson JO, Sicheritz-PontenT, Alsmark UCM, Podowski RM, et al. The genome sequence ofRickettsia prowazekii and the origin of mitochondria. Nature 1998;396: 133-43.

[54] Segal G, Shuman HA. How is the intracellular fate of theLegionella pneumophila phagosome determined? Trends inMicrobiol 1998; 6: 253-5.

[55] Cianciotto NP. Pathogenicity of Legionella pneumophila. Int J MedMicrobiol 2001; 291: 331-43.

[56] Hobbs M, Mattick JS. Common components in the assembly oftype 4 fimbriae, DNA transfer systems, filamentous phage andprotein-secretion apparatus: a general system for the formation ofsurface-associated protein complexes. Mol Microbiol 1993; 10:233-43.

[57] Zink SD, Pedersen L, Cianciotto NP, Abu-Kwaik Y. The Dot/Icmtype IV secretion system of Legionella pneumophila is essential forthe induction of apoptosis in human macrophages. Infect Immun2002; 70: 1657-63.

[58] Nagai H, Roy CR. The DotA protein from Legionella pneumophilais secreted by a novel process that requires the Dot/Icm transporter.EMBO J 2001; 20: 5962-70.

[59] Vogel JP, Andrews HL, Wong SK, Isberg RR. Conjugative transferby the virulence system of Legionella pneumophila. Science 1998;279: 873-6.

[60] Segal G, Purcell M, Shuman HA. Host cell killing and bacterialconjugation require overlapping sets of genes within a 22-kb regionof the Legionella pneumophila genome. Proc Natl Acad Sci USA1998; 95: 1669-74.

[61] Segal G, Russo JJ, Shuman HA. Relationships between a new typeIV secretion system and the icm/dot virulence system of Legionellapneumophila. Mol Microbiol 1999; 34: 709-809.

[62] Felek S, Huang H, Rikihisa Y. Sequence and expression analysis ofvirB9 of the type IV secretion system of Ehrlichia canis strains inticks, dogs, and cultured cells. Infect Immun 2003; 71: 6063-7.

[63] Economou A. Following the leader: bacterial protein exportthrough the Sec pathway. Trends Microbiol 1999; 7: 315-20.

[64] Thanassi DG, Hultgren SJ. Multiple pathways allow proteinsecretion across the bacterial outer membrane. Curr Opin Cell Biol2000; 12: 420-30.

[65] Cheng KS, Lu MC, Tang HL, Chou FT. Phosphorylation ofHelicobacter pylori CagA in patients with gastric ulcer andgastritis. Adv Ther 2002; 19: 85-90.

[66] Marshall B. Helicobacter pylori: 20 years on. Clin Med 2002; 2:147-52.

[67] Christie PJ. The cag pathogenicity island: mechanistic insights.Trends in Microbiol 1997; 5: 264-5.

[68] Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W, Haas R.Translocation of Helicobacter pylori CagA into gastric epithelialcells by type IV secretion. Science 2000; 287: 1497-500.

[69] Backert S, Churin Y, Meyer TF, Helicobacter pylori type IVsecretion, host cell signalling and vaccine development. Keio JMed 2002; 51 (Suppl. 2): 6-14.

[70] Kuipers EJ, Israel DA, Kusters JG, Blaser MJ. Evidence for aconjugation-like mechanism of DNA transfer in Helicobacterpylori. J Bacteriol 1998; 180: 2901-5.

[71] Smeets LC, Kusters JG. Natural transformation in Helicobacterpylori: DNA transport in an unexpected way. Trends Microbiol2002; 10: 159-62.

[72] Hofreuter D, Odenbreit S, Haas R. Natural transformationcompetence in Helicobacter pylori is mediated by the basic

components of a type IV secretion system. Mol Microbiol 2001;41: 379-91.

[73] Suerbaum S, Josenhans C, Sterzenbach T, Drescher B, Brandt P,Bell M, et al. The complete genome sequence of the carcinogenicbacterium Helicobacter hepaticus . Proc Natl Acad Sci USA 2003;100: 7901-6.

[74] Strauch E, Goelz G, Knabner D, Konietzny A, Lanka E, Appel B.A cryptic plasmid of Yersinia enterocolitica encodes a conjugativetransfer system related to the regions of CloDF13 Mob and IncXPil. Microbiology 2003; 149: 2829-45.

[75] Weiss AA, Johnson FD, Burns DL. Molecular characterization ofan operon required for pertussis toxin secretion. Proc Natl Acad SciUSA 1993; 90: 2970-4.

[76] Cabezón E, Sastre JI, de la Cruz F. Genetic evidence of a couplingrole for the TraG protein family in bacterial conjugation. Mol GenGenet 1997; 254: 400-6.

[77] Llosa M, Gomis-Rüth FX, Coll M, de la Cruz F. Bacterialconjugation: a two-step mechanism for DNA transport. MolMicrobiol 2002; 45: 1-8.

[78] Selbach M, Moese S, Meyer TF, Backert S. Functional analysis ofthe Helicobacter pylori cag pathogenicity island reveals bothVirD4-CagA-dependent and VirD4-CagA-independentmechanisms. Infect Immun 2002; 70: 665-71.

[79] Kumar RB, Das A. Polar location and functional domains of theAgrobacterium tumefaciens DNA transfer protein VirD4. MolMicrobiol 2002; 43: 1523-32.

[80] Okamoto S, Toyoda-Yamamoto A, Ito K, Takebe I, Machida Y.Localization and orientation of the VirD4 protein of Agrobacteriumtumefaciens in the cell membrane. Mol Gen Genet 1991; 228: 24-32.

[81] Panicker MM, Minkley EG. Purification and properties of the F sexfactor TraD protein, an inner membrane conjugal transfer protein. JBiol Chem 1992; 267: 12761-6.

[82] Moncalián G, Cabezón E, Alkorta I, Valle M, Moro F, ValpuestaJM, et al. Characterization of ATP and DNA binding activities ofTrwB, the coupling protein essential in plasmid R388 conjugation.J Biol Chem 1999; 274: 36117-24.

[83] Balzer D, Pansegrau W, Lanka E. Essential motifs of relaxase(TraI) and TraG proteins involved in conjugative transfer ofplasmid RP4. J Bacteriol 1994; 176: 4285-95.

[84] Lessl M, Pansegrau W, Lanka E. Relationship of DNA-transfersystems: essential transfer factors of plasmids RP4, Ti and F sharecommon sequences. Nucl Acids Res 1992; 20: 6099-100.

[85] Llosa M, Bolland S, de la Cruz F. Genetic organization of theconjugal DNA processing region of the IncW plasmid R388. J MolBiol 1994; 235: 448-64.

[86] Das A, Xie Y-H. Construction of transposon Tn3phoA: itsapplication in defining the membrane topology of theAgrobacterium tumefaciens DNA transfer proteins. Mol Microbiol1998; 27: 405-14.

[87] Lee MH, Kosuk N, Bailey J, Traxler B, Manoil C. Analysis of Ffactor TraD membrane topology by use of gene fusions andtrypsin-sensitive insertions. J Bacteriol 1999; 181: 6108-13.

[88] Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly relatedsequences in the α- and β-subunits of ATP synthase, myosin,kinases and other ATP-requiring enzymes and a commonnucleotide binding fold. EMBO J 1982; 1: 945-51.

[89] Santini JM, Stanisch VA. Both the fipA gene of pKM101 and thepifC gene of F inhibit conjugal transfer of RP1 by an effect ontraG. J Bacteriol 1998; 180: 4093-101.

[90] Berger BR, Christie PJ. Genetic complementation analysis of theAgrobacterium tumefaciens virB operon: virB2 through virB11 areessential virulence genes. J Bacteriol 1994; 176: 3646-60.

[91] Lessl M, Balzer D, Pansegrau W, Lanka E. Sequence similaritiesbetween the RP4 Tra2 and the Ti VirB region strongly support theconjugation model for T-DNA transfer. J Biol Chem 1992; 267:20471-80.

[92] Szpirer CY, Faelen M, Couturier M. Interaction between the RP4coupling protein TraG and the pBHR1 mobilization protein Mob.Mol Microbiol 2000; 37: 1283-92.

[93] Cabezón E, Lanka E, de la Cruz F. Requirements for mobilizationof plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwBand RP4 traG are interchangeable. J Bacteriol 1994; 176: 4455-8.

[94] Hamilton CM, Lee H, Li P-L, Cook DM, Piper KR, Beck vonBodman S, et al. TraG from RP4 and TraG and VirD4 from Ti


plasmids confer relaxosome specificity to the conjugal transfersystem of pTiC58. J Bacteriol 2000; 182: 1541-8.

[95] Sastre JI, Cabezon E, de la Cruz F. The carboxyl terminus ofprotein TraD adds specificity and efficiency to F-plasmidconjugative transfer. J Bacteriol 1998; 180: 6039-42.

[96] Karplus K. Hidden Markov models for detecting remote proteinhomologies. Bioinformatics 1998; 14: 846-56.

[97] Schröder G, Krause S, Zechner EL, Traxler B, Yeo HJ, Lurz R,et al. TraG-like proteins of DNA transfer systems and of theHelicobacter pylori type IV secretion system: inner membrane gatefor exported substrates? J Bacteriol 2002; 184: 2767-79.

[98] Disque-Kochem C, Dreiseikelmann B. The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD invitro. J Bacteriol 1997; 179(19): 6133-7.

[99] Gilmour MW, Gunton JE, Lawley TD, Taylor DE. Interactionbetween the IncHI1 plasmid R27 coupling protein and type IVsecretion system: TraG associates with the coiled-coil mating pairformation protein TrhB. Mol Microbiol 2003; 49: 105-16.

[100] Bath J, Wu LJ, Errington J, Wang JC. Role of Bacillus subtilisSpoIIIE in DNA transport across the mother cell-prespore divisionsepyum. Science 2000; 290: 995-7.

[101] Wu LJ, Lewis PJ, Allmansberger R, Hauser PM, Errington J. Aconjugation-like mechanism for prespore chromosome partitioningduring sporulation in bacillus subtilis. Gen Dev 1995; 9: 1316-26.

[102] Errington J, Bath J, Wu LJ. DNA transport in bacteria. Nat RevMol Cell Biol 2001; 2: 538-45.

[103] Sharp MD, Pogliano K. Role of cell-specific SpoIIIE assembly inpolarity of DNA transfer. Science 2002; 295: 137-9.

[104] Wu LJ, Errington J. Septal localization of the SpoIIIE chromosomepartitioning protein in Bacillus subtilis. EMBO J 1997; 16: 2161-9.

[105] Recchia GD, Aroyo M, Wolf D, Blakely G, Sherratt DJ. FtsK-dependent and -independent pathways of Xer site-specificrecombination. EMBO J 1999; 18: 5724-34.

[106] Steiner W, Liu GW, Donachie WD, Kuempel P. The cytoplasmicdomain of FtsK protein is required for resolution of chromosomedimers. Mol Microbiol 1999; 31: 579-83.

[107] Datta N, Hedges RW. Trimethoprim resistance conferred by Wplasmids in Enterobacteriaceae. J Gen Microbiol 1972; 72: 349-55.

[108] Swift G, McCarthy BJ, Heffron F. DNA sequence of plasmid-encoded dihydrofolat reductase. Mol Gen Genet 1981; 181: 441-7.

[109] Swedberg G, Skold O. Plasmid-borne sulfonamide resistancedeterminants studied by restriction enzyme analysis. J Bacteriol1983; 153: 1228-37.

[110] Bolland S, Llosa M, Avila P, de la Cruz F. General organization ofthe conjugal transfer genes of the IncW plasmid R388 andinteractions between R388 and IncN and IncP plasmids. J Bacteriol1990; 172: 5795-802.

[111] Guasch A, Lucas M, Cabezas M, Pérez-Luque R, Gomis-Rüth FX,de la Cruz F, et al. Recognition and processing of the origin oftransfer DNA by conjugative relaxase TrwC. Nat Struct Biol 2003;10, 1002-1010.

[112] Llosa M, Grandoso G, de la Cruz F. Nicking activity of TrwCdirected against the origin of transfer of the IncW plasmid R388. JMol Biol 1995; 246: 54-62.

[113] Grandoso G, Llosa M, Zabala JC, de la Cruz F. Purification andbiochemical characterization of TrwC, the helicase involved inplasmid R388 conjugal DNA transfer. Eur J Biochem 1994; 226:403-12.

[114] Moncalián G, Grandoso G, Llosa M, de la Cruz F. oriT-processingand regulatory roles of TrwA protein in plasmid R388 conjugation.J Mol Biol 1997; 270: 188-200.

[115] Rice PA, Yang S-W, Mizuuchi K, Nash HA. Crystal structure of anIHF-DNA complex: a protein induced DNA U-turn. Cell 1996; 87:1295-306.

[116] Moncalián G, Valle M, Valpuesta JM, de la Cruz F. IHF proteininhibits cleavage but not assembly of plasmid R388 relaxosomes.Mol Microbiol 1999; 31: 1643-52.

[117] Gomis-Rüth FX, Moncalián G, Pérez-Luque R, González A,Cabezón E, de la Cruz F, et al. The bacterial conjugation proteinTrwB resembles ring helicases and F1-ATPase. Nature 2001; 409:637-41.

[118] Gomis-Rüth FX, Moncalián G, de la Cruz F, Coll M. Conjugativeplasmid protein TrwB, an integral membrane type IV secretionsystem coupling protein: detailed structural features and mappingof the active-site cleft. J Biol Chem 2002; 277: 7556-66.

[119] Dalal S, Hanson PI. Membrane traffic: what drives the AAAmotor? Cell 2001; 104: 5-8.

[120] Vale RD. AAA proteins: lords of the ring. J Cell Biol 2000; 150:F13-F9.

[121] Guenther B, Onrust R, Sali A, O'Donnell M, Kuriyan J. Crystalstructure of the δ' subunit of the clamp-loader complex of E.coliDNA polymerase III. Cell 1997; 91: 335-45.

[122] Lenzen CU, Steinmann D, Whiteheart SW, Weis WI. Crystalstructure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell 1998; 94: 525-35.

[123] Zhang X, Shaw A, Bates PA, Newman RH, Gowen B, Orlova E,et al. Structure of the AAA ATPase p97. Mol Cell 2000; 6: 1473-84.

[124] Bochtler M, Hartmann C, Song HK, Bourenkov GP, Bartunik HD,Huber R. The structures of HsIU and the ATP-dependent proteaseHsIU-HsIV. Nature 2000; 403: 800-5.

[125] Waksman G, Lanka E, Carazo J-M. Helicases as nucleic acidunwinding machines. Nat Struct Biol 2000; 7: 20-2.

[126] Soultanas P, Wigley DB. DNA helicases: 'inching forward'. CurrOp Struct Biol 2000; 10: 124-8.

[127] Sawaya MR, Guo S, Tabor S, Richardson CC, Ellenberger T.Crystal structure of the helicase domain from the replicativehelicase-primase of bacteriophage T7. Cell 1999; 99: 167-77.

[128] Hacker KJ, Johnson KA. A hexameric helicase encircles one DNAstrand and excludes the other during DNA unwinding.Biochemistry 1997; 36: 14080-7.

[129] Schwer B. A new twist on RNA helicases: DExxH/D box proteinsas RNPases. Nat Struct Biol 2001; 8: 113-6.

[130] Singleton MR, Sawaya MR, Ellenberger T, Wigley DB. Crystalstructure of T7 gene 4 ring helicase indicates a mechanism forsequential hydrolysis of nucleotides. Cell 2000; 101: 589-600.

[131] Niedenzu T, Röleke D, Bains G, Scherzinger E, Saenger W.Crystal structure of the hexameric replicative helicase RepA ofplasmid RSF1010. J Mol Biol 2001; 306: 479-87.

[132] Yu X, Egelman EH. The RecA hexamer is a structural homologueof ring helicases. Nat Struct Biol 1997; 4(2): 101-4.

[133] Story RM, Weber IT, Steitz TA. The structure of the E.coli recAprotein monomer and polymer. Nature 1992; 355: 318-25.

[134] Story RM, Steitz TA. Structure of the recA protein - ADP complex.Nature 1992; 355: 374-6.

[135] Subramanya HS, Bird LE, Brannigan JA, Wigley DB. Crystalstructure of a DExx box DNA helicase. Nature 1996; 384: 379-83.

[136] Velankar SS, Soultanas P, Dillingham MS, Subramanya HS,Wigley DB. Crystal structures of complexes of PcrA DNA helicasewith a DNA substrate indicate an inchworm mechanism. Cell 1999;97: 75-84.

[137] Machón C, Rivas S, Albert A, Goñi FM, de la Cruz F. TrwD, thehexameric traffic ATPase encoded by plasmid R388, inducesmembrane destabilization and hemifusion of lipid vesicles. JBacteriol 2002; 184: 1661-8.

[138] Yeo H-J, Savvides SN, Herr AB, Lanka E, Waksman G. Crystalstructure of the hexameric traffic ATPase of the Helicobacterpylori type IV secretion system. Mol Cell 2000; 6: 1461-72.

[139] Subramanya HS, Arciszewska LK, Baker RA, Bird LE, SherrattDJ, Wigley DB. Crystal structure of the site-specific recombinase,XerD. EMBO J 1997; 16: 5178-87.

[140] Stockner T, Plugariu C, Koraimann G, Högenauer G, Bermel W,Prytulla S, et al. Solution structure of the DNA-binding domain ofTraM. Biochemistry 2001; 40: 3370-7.

[141] Kupelwieser G, Schwab M, Hogenauer G, Koraimann G, ZechnerEL. Transfer protein TraM stimulates TraI-catalyzed cleavage ofthe transfer origin of plasmid R1 in vivo. J Mol Biol 1998; 275:81-94.

[142] Yu X, Shibata T, Egelman EH. Identification of a defined epitopeon the surface of the active RecA-DNA filament using amonoclonal antibody and three-dimensional reconstruction. J MolBiol 1998; 283: 985-92.

[143] Abrahams JP, Leslie AGW, Lutter R, Walker JE. Structure at 2.8 Åresolution of F1-ATPase from bovine heart mitochondria. Nature1994; 370: 621-8.

[144] Montallebi-Veshareh M, Balzer D, Lanka E, Jagura-Burdzy G,Thomas CM. Conjugative transfer functions of broad-host-rangeplasmid RK2 are coregulated with vegetative replication. MolMicrobiol 1992; 6: 907-20.

[145] Savvides SN, Yeo HJ, Beck MR, Blaesing F, Lurz R, Lanka E,et al. VirB11 ATPases are dynamic hexameric assemblies: new


insights into bacterial type IV secretion. EMBO J 2003; 22: 1969-80.

[146] Rivas S, Bolland S, Cabezón E, Goñi FM, de la Cruz F. TrwD, aprotein encoded by the IncW plasmid R388, displays an ATPhydrolase activity essential for bacterial conjugation. J Biol Chem1997; 272: 25583-90.

[147] Parsons CA, Stasiak A, Bennett RJ, West SC. Structure of amultisubunit complex that promotes DNA branch migration.Nature 1995; 374: 375-8.

[148] Reinisch KM, Nibert ML, Harrison SC. Structure of the reoviruscore at 3.6 Å resolution. Nature 2000; 404: 960-7.

[149] Simpson AA, Tao Y, Leiman PG, Badasso MO, He Y, Jardine PJ,et al. Structure of the bacteriophage Φ29 DNA packaging motor.Nature 2000; 408: 745-50.

[150] Guasch A, Pous J, Ibarra B, Gomis-Rüth FX, Valpuesta JM, SousaN, et al. Detailed architecture of a DNA translocating machine: thehigh-resolution structure of the bacteriophage Φ29 connectorparticle. J Mol Biol 2002; 315(4): 663-76.

[151] Egelman EH, Yu X, Wild R, Hingorani MM, Patel SM.Bacteriophage T7 helicase/primase proteins form rings aroundsingle-strandd DNA that suggest a general structure for hexamerichelicases. Proc Natl Acad Sci USA 1995; 92: 3869-73.

[152] Hingorani MM, O'Donnell M. Toroidal proteins: Running ringsaround DNA. Curr Biol 1998; 8: R83-R6.

[153] Sullivan JT, Trzebiatowski JR, Cruickshank RW, Gouzy J, BrownSD, Elliot RM, et al. Comparative sequence analysis of thesymbiosis island of Mesorhizobium loti strain R7A. J Bacteriol2002; 184: 3086-95.

[154] Bittinger MA, Gross JA, Widom J, Clardy J, Handelsman J.Rhizobium etli CE3 carries vir gene homologs on a self-transmissible plasmid. Mol Plant Microbe Interact 2000; 13: 1019-21.

[155] Segal G, Shuman HA. Possible origin of the Legionellapneumophila virulence genes and their relation to Coxiella burnetii.Mol Microbiol 1999; 33: 669-70.

[156] Padmalayam I, Karem K, Baumstark B, Massung R. The geneencoding the 17-kDa antigen in Bartonella henselae is locatedwithin a cluster of genes homologous to the virB virulence operon.DNA Cell Biol 2000; 19: 377-82.

[157] Baron C, O'Callaghan D, Lanka E. Bacterial secrets of secretion:EuroConference on the biology of type IV secretion processes. MolMicrobiol 2002; 43: 1359-65.

[158] Seubert A, Schulein R, Dehio C. Bacterial persistence withinerythrocytes: a unique pathogenic strategy of Bartonella spp. Int JMed Microbiol 2002; 291: 555-60.

[159] Seubert A, Hiestand R, de la CF, Dehio C. A bacterial conjugationmachinery recruited for pathogenesis. Mol Microbiol 2003; 49:1253-66.

[160] Schmiederer M, Arcenas R, Widen R, Valkov N, Anderson B.Intracellular induction of the Bartonella henselae virB operon byhuman endothelial cells. Infect Immun 2001; 69: 6495-502.

[161] Bacon DJ, Alm RA, Burr DH, Hu L, Kopecko DJ, Ewing CP, et al.Involvement of a plasmid in virulence of Campylobacter jejuni 81-176. Infect Immun 2000; 68: 4384-90.

[162] Bacon DJ, Alm RA, Hu L, Hickey TE, Ewing CP, Batchelor RA,et al. DNA sequence and mutational analyses of the pVir plasmidof Campylobacter jejuni 81-176. Infect Immun 2002; 70: 6242-50.

[163] Novak KF, Dougherty B, Peláez M. Actinobacillus actinomycetem-comitans harbours type IV secretion system genes on a plasmid andin the chromosome. Microbiology 2001; 147: 3027-35.

[164] Henderson B, Nair SP, Ward JM, Wilson M. Molecularpathogenicity of the oral opportunistic pathogen Actinobacillusactinomycetemcomitans. Annu Rev Microbiol 2003; 57: 29-55.

[165] Sexton JA, Vogel JP. Type IVB secretion by intracellularpathogens. Traffic 2002; 3: 178-85.

[166] Zamboni DS, McGrath S, Rabinovitch M, Roy CR. Coxiellaburnetii express type IV secretion system proteins that functionsimilarly to components of the Legionella pneumophila Dot/Icmsystem. Mol Microbiol 2003; 49: 965-76.

[167] Stouthamer R, Breeuwer JAJ, Hurst GDD. Wolbachia pipientis:microbial manipulator of arthropod reproduction. Annu RevMicrobiol 1999; 53: 71-102.

[168] Masui S, Sasaki T, Ishikawa H. Genes for the type IV secretionsystem in an intracellular symbiont, Wolbachia, a causative agentof various sexual alterations in arthropodes. J Bacteriol 2000; 182:6529-31.

[169] Liu H, Kang Y, Genin S, Schell MA, Denny TP. Twitchingmotility of Ralstonia solanacearum requires a type IV pilus system.Microbiology 2001; 147: 3215-29.

[170] Jones CS, Osborne DJ. Identification of contemporary plasmidvirulence genes in ancestral isolates of Salmonella enteritidis andSalmonella typhimurium. FEMS Microbiol Lett 1991; 64: 7-11.

[171] da Silva ACR, Ferro JA, Reinach FC, Farah CS, Furlan LR,Quaggio RB, et al. Comparison of the genomes of twoXanthomonas pathogens with differing host specificities. Nature2002; 417: 459-63.

[172] Yoshioka Y, Fujita Y, Ohtsubo E. Nucleotide sequence of thepromoter-distal region of the tra operon of plasmid R100, includingtraI (DNA helicase I) and traD genes. J Mol Biol 1990; 214: 39-53.

[173] Jalajakumari MB, Manning PA. Nucleotide sequence of the traDregion in the Escherichia coli F sex factor. Gene 1989; 81: 195-202.

[174] Romine MF, Stillwell LC, Wong KK, Thurston SJ, Sisk EC,Sensen C, et al. Complete sequence of a 184-kilobase catabolicplasmid from Sphingomonas aromaticivorans F199. J Bacteriol1999; 181: 1585-602.

[175] Coupland GM, Brown AM, Willetts NS. The origin of transfer(oriT) of the conjugative plasmid R46: characterization by deletionanalysis and DNA sequencing. Mol Gen Genet 1987; 208: 219-25.

[176] Paterson ES, Moré MI, Pillay G, Cellini C, Woodgate R, WalkerGC, et al. Genetic analysis of the mobilization and leading regionsof the IncN plasmids pKM101 and pCU1. J Bacteriol 1999; 181:2572-83.

[177] Hirayama T, Muranaka T, Ohkawa H, Oka A. Organization andcharacterization of the virCD genes from Agrobacteriumrhizogenes. Mol Gen Genet 1988; 213: 229-37.

[178] Porter SG, Yanofsky MF, Nester EW. Molecular characterizationof the virD operon from Agrobacterium tumefaciens. Nucleic AcidsRes 1987; 15: 7503-17.

[179] Rogowsky PM, Powell BS, Shirasu K, Lin TS, Morel P, ZyprianEM, et al. Molecular characterization of the vir regulon ofAgrobacterium tumefaciens: complete nucleotide sequence andgene organization of the 28.63-kbp regulon cloned as a single unit.Plasmid 1990; 23: 85-106.

[180] Ogata H, Audic S, Renesto-Audiffren P, Fournier P-E, Barbe V,Samson D, et al. Mechanisms of evolution in Rickettsia conorii andR. prowazekii. Science 2001; 293: 2093-8.

[181] Ziegelin G, Pansegrau W, Strack B, Balzer D, Kröger M, Kruft V,et al. Nucleotide sequence and organization of genes flanking thetransfer origin of promiscuous plasmid RP4. DNA Sequence 1991;1: 303-27.

[182] Komano T, Kubo A, Nisioka T. Shufflon: multi-inversion of fourcontiguous DNA segments of plasmid R64 creates seven differentopen reading frames. Nucleic Acids Res 1987; 15: 1165-72.

[183] Tomb J-F, White O, Kerlavage AR, Clayton RA, Sutton GG,Fleischmann RD, et al. The complete genome sequence of thegastric pathogen Helicobacter pylori. Nature 1997; 388: 539-47.

[184] Simpson AJG, Reinach FC, Arruda P, Abreu FA, Acencio M,Alvarenga R, et al. The genome sequence of the plant pathogenXylella fastidiosa. Nature 2000; 406: 151-157.

[185] Galli DM, Chen J, Novak KF, Leblanc DJ. Nucleotide sequenceand analysis of conjugative plasmid pVT745. J Bacteriol 2001;183: 1585-94.

[186] Freiberg C, Fellay R, Bairoch A, Broughton WJ, Rosenthal A,Perret X. Molecular basis of symbiosis between Rhizobium andlegumes. Nature 1997; 387: 394-401.

[187] Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S,et al. Complete genome structure of the nitrogen-fixing symbioticbacterium Mesorhizobium loti. DNA Res 2000; 7: 331-8.

[188] Alt-Morbe J, Stryker JL, Fuqua C, Li PL, Farrand SK, Winans SC.The conjugal transfer system of Agrobacterium tumefaciensoctopine-type Ti plasmids is closely related to the transfer systemof an IncP plasmid and distantly related to Ti plasmid vir genes. JBacteriol 1996; 178: 4248.

[189] Farrand SK, Hwang I, Cook DM. The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfersystems of RSF1010, RP4, and F. J Bacteriol 1996; 178: 4233-47.

[190] Salanoubat M, Genin S, Artiguenave F, Gouzy J, Mangenot S,Arlat M, et al. Genome sequence of the plant pathogen Ralstoniasolanacearum. Nature 2002; 415: 497-502.


[191] Alarcón-Chaídez F, Sampath J, Srinivas P, Vijayakumar MN.Tn5252: a model for complex streptococcal conjugativetransposons. Adv Exp Med Biol 1997; 418: 1029-32.

[192] Noelling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q,Gibson R, et al. Genome sequence and comparative analysis of thesolvent-producing bacterium Clostridium acetobutylicum. JBacteriol 2001; 183: 4823-38.

[193] Schneiker S, Keller M, Droge M, Lanka E, Puhler A, SelbitschkaW. The genetic organization and evolution of the broad host rangemercury resistance plasmid pSB102 isolated from a microbialpopulation residing in the rhizosphere of alfalfa. Nucl Acids Res2001; 29: 5169-81.

[194] Morton TM, Eaton DM, Johnston JL, Archer GL. DNA sequenceand units of transcription of the conjugative transfer gene complex(trs) of Staphylococcus aureus plasmid pGO1. J Bacteriol 1993;175: 4436-47.

[195] Dougherty BA, Hill C, Weidman JF, Richardson DR, Venter JC,Ross RP. Sequence and analysis of the 60 kb conjugativebacteriocin-producing plasmid pMRC01 from Lactococcus lactisDPC3147. Mol Microbiol 1998; 29: 1029-38.

[196] Garnier F, Taourit S, Glaser P, Courvalin P, Galimand M.Characterization of transposon Tn1549, conferring VanB-typeresistance in Enterococcus spp.. Microbiology 2000; 146: 1481-9.

[197] Nuñez B, Ávila P, de la Cruz F. Genes involved in conjugativeDNA processing of plasmid R6K. Mol Microbiol 1997; 24: 1157-68.

[198] Franco AA, Cheng R, Chung G-T, Wu S, Sears CL. Molecularevolution of the pathogenicity island of enterotoxigenicBacteroides fragilis strains. J Bacteriol 1999; 18: 6623-33.

[199] van Putten AJ, Jochems GJ, de Lang R, Nijkamp HJJ. Structureand nucleotide sequence of the region encoding the mobilizationproteins of plasmid CloDF13. Gene 1987; 51: 171-8.

[200] Giacomini A, Squartini A, Nuti MP. Nucleotide sequence andanalysis of plasmid pMD136 from Pediococcus pentosaceusFBB61 (ATCC43200) involved in Pediocin A production. Plasmid2000; 43: 111-22.

[201] Tauch A, Schneiker S, Selbitschka W, Puhler A, Van Overbeek LS,Smalla K, et al. The complete nucleotide sequence andenvironmental distribution of the cryptic, conjugative, broad-host-

range plasmid pIPO2 isolated from bacteria of the wheatrhizosphere. Microbiology 2002; 148: 1637-53.

[202] Godon JJ, Pillidge CJ, Jury K, Gasson MJ. Characterization of anoriginal conjugative element - the sex factor of Lactococcus lactis712. Lait 1996; 76: 41-9.

[203] Takai S, Hines SA, Sekizaki T, Nicholson VM, Alperin DA, OsakiM, et al. DNA sequence and comparison of virulence plasmidsfrom Rhodococcus equi ATCC 33701 and 103. Infect Immun 2000;68: 6840-7.

[204] Okinaka R, Cloud K, Hampton O, Hoffmaster A, Hill K, Keim P,et al. Sequence, assembly and analysis of pX01 and pX02. J ApplMicrobiol 1999; 87: 261-2.

[205] Stedman KM, She Q, Phan H, Holz I, Singh H, Prangishvili D,et al. pING family of conjugative plasmids from the extremelythermophilic archaeon Sulfolobus islandicus: insights intorecombination and conjugation in crenarchaeota. J Bacteriol 2000;182: 7014-20.

[206] She Q, Phan H, Garrett RA, Albers SV, Stedman KM, Zillig W.Genetic profile of pNOB8 from Sulfolobus: the first conjugativeplasmid from an archaeon. Extremophiles 1998; 2: 417-25.

[207] Cloeckaert A, Baucheron S, Chaslus-Dancla E. Nonenzymaticchloramphenicol resistance mediated by IncC plasmid R55 isencoded by a floR gene variant. Antimicrob Agents Chemother2001; 45: 2381-2.

[208] Begg KJ, Dewar SJ, Donachie WD. A new Escherichia coli celldivision gene, ftsK. J Bacteriol 1995; 177: 6211-22.

[209] Krall L, Wiedemann U, Unsin G, Weiss S, Domke N, Baron C.Detergent extraction identifies different VirB protein subassembliesof the type IV secretion machinery in the membranes ofAgrobacterium tumefaciens. Proc Natl Acad Sci USA 2002; 99:11405-10.

[210] Frankel AE, Koo HM, Leppla SH, Duesbery NS. and Wounde GF.Novel protein targeted therapy of metastatic melanoma. CurrPharm Des 2003; 9(25): 2060-6.

[211] Ibrahim HR, Aoki T. and Pellegrini A. Strategies for newantimicrobial proteins and peptides: lysozyme and aprotinin asmodel molecules. Curr Pharm Des 2002; 8(9): 671-93.

[212] Ilies MA, Seitz WA. and Balaban AT. Cationic lipids in genedelivery: principles, vector design and therapeutical applications.Curr Pharm Des 2002; 8(27): 2441-73.

Coupling Factors in Macromolecular Type-IV Secretion Machineries

Documents

Transcript of Coupling Factors in Macromolecular Type-IV Secretion Machineries