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-Rüth 1, * , M. Solà 1 , F. de la Cruz 2 and M. Coll 1, * 1 Institut de Biologia Molecular de Barcelona, C.S.I.C., c/ Jordi Girona, 18-26, E-08034 Barcelona (Spain) and 2 Departamento de Biología Molecular (Laboratorio asociado al C.I.B., C.S.I.C.), Universidad de Cantabria, c/ Herrera Oria, 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 formation in plant cells, as observed in Agrobacterium, toxin secretion, like in Bordetella and Helicobacter, cell-to-cell translocation of virulence factors, and intracellular activity of mammalian pathogens like Legionella. By enabling conjugative DNA delivery, these systems contribute to the spread of antibiotic resistance genes among bacteria. These translocons are made up by 10-15 proteins that are analogous to Vir proteins of Agrobacterium and traverse both membranes and the periplasmic 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, the multimeric type-IV coupling proteins (T4CPs), to connect the macromolecular complexes to be transferred with the secretory conduit. To do so, these T4CPs may be required to localise close to the secretion machinery within the donor cell. The T4CP structural prototype is the hexameric protein TrwB of Escherichia coli conjugative plasmid R388, closely related to Agrobacterium VirD4 protein. It is responsible for coupling the relaxosome with the DNA transport apparatus during cell mating. T4CP family members are related to SpoIIIE/FtsK proteins, essential for DNA pumping during sporulation and cell division. These features suggest possible mechanisms for conjugal T4CP function: as a simple coupler between two molecular machines, as a rotating device to pump DNA through the type-IV transport pore, or as a DNA 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-dimensional structure. TYPE-IV SECRETION, BACTERIAL CONJUGATION AND PATHOGENESIS Bacteria are not solely dependent on their internal milieu to accomplish their living strategies. They often secrete enzymes and other macromolecules to the external medium in order to readapt it according to their own requirements. Secretion machineries spanning the cell membrane(s) are responsible for macromolecular transport. These systems can be classified into five types (I to V) and share the presence of proteins using nucleosyl triphosphate (NTP) hydrolysis as a transport driving force, except for the autotransporter system, which does not depend on NTP [1-10]. Over a dozen distinct families of protein translocating systems, mostly restricted to bacteria, have been characterised and classified [11, 12]. A sophisticated variant of macromolecular secretion is type-IV translocation, through which (nucleo)proteins can be directly injected into prokaryotic and eukaryotic recipient cells. In an extreme version, DNA is translocated to the recipient cell, so that its metabolism is permanently altered. The type-IV pathway encompasses a multiprotein bacterial transmembrane organelle or translocon specialised in the intercellular transfer of a variety of macromolecules. It features a filamentous macromolecular apparatus protruding *Address correspondence to these athors at the Institut de Biologia Molecular de Barcelona, C.S.I.C., c/ Jordi Girona, 18-26, E-08034 Barcelona (Spain); Tel: +3493 400 61 44/49; Fax: +3493 204 59 04; E-mail: [email protected] and [email protected]. from the bacterial envelope. T4SSs can be controlled by transcriptional repressor systems, like KorA/KorB, which regulate partitioning genes [13]. It differs from the other four systems in that it is conjugation-related or an “adapted conjugation” system, capable of DNA translocation [11, 14, 15]. The substrates are as diverse as nucleoprotein particles and multicomponent protein associations that cross the bacterial 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 substrates can also occur in the cytoplasm, the inner membrane or the periplasm of the bacterial cell [1]. These translocons typically consist of 10-15 proteins, encoded by a single gene cluster, and they form two major structural components: an extracellular filamentous surface appendage or sex pilus, for initiating physical contact between the donor and recipient cells [18], and a membrane-associated protein complex that translocates substrates across the membranes involved [19]. It is still a matter of debate whether the pilus belongs to the conjugative pore or merely brings cells together. Since the pilus is not present in Gram-positive bacteria, it does not seem to be indispensable. This pathway is involved in bacterial conjugation, a contact-dependent process often induced by extracellular or growth-phase dependent signals [20-23]. It is mediated by conjugative plasmids or transposons that transfer DNA unidirectionally from a donor to a recipient cell across the donor cell envelope, rendering a recipient that may become a potential donor (Fig. (1a); [21, 24]). It is a means for rapid evolution, by which bacterial populations adapt to a changing environment, e.g. acquisition of resistance to antibiotics. It is also a broadly applicable tool to shuttle

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Page 1: 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.


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

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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

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[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).



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]

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1554 Current Pharmaceutical Design, 2004, Vol. 10, No. 13 Gomis-Rüth et al.

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 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 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

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Coupling Factors in Macromolecular Type-IV Secretion Machineries Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1555



Data Bank access code

(GB, GenBank; SP, SwissProt/

TrEMBL; PIR, Prot.Inf.Res.)





Escherichia coli R388

Xanthomonas axonopodis pXAC64

E. coli R100

E. coli F

Sphyngomonas aromaticivorans pNL1



SP Q04230


SP P22708

SP O87742, P09130

SP 085919

SP O06958












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 Q00185

SP Q00184

GB AB027308



















Hypot. Protein SCE2.07



VirD4-like protein


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


Pediococcus pentosaceus pMD136

X. axonopodis


E. coli pIPO2T

Anaplasma phagocytophylum

Ehrlichia chaffeensis

R. loti

X. campestris

L. lactis 712

SP O25252


SP Q9F254

SP Q9F531

SP P55421

SP Q98NT6, Q981R7, GB CAD31437

SP Q44346

GB NP_066690


SP Q937B8

GB AAG38037




SP Q07713

SP O87219

GB AAF72343

SP O32582

SP O25260, Q9ZLV3, Q9JMY0, GB


PIR T18329, SP O54524


SP P08098

PIR T36167

GB NP_052502


GB AAM37472

GB AAM41759, AAM42732




GB CAD31465





direct submission




direct submission


direct submission


direct submission











direct submission

direct submission








direct submission


(Table 2) contd….

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1556 Current Pharmaceutical Design, 2004, Vol. 10, No. 13 Gomis-Rüth et al.



Data Bank access code

(GB, GenBank; SP, SwissProt/

TrEMBL; PIR, Prot.Inf.Res.)


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


gnl|WUGSC 99287|stmlt2-.Contig1522

gnl|CUCGC 446|lpneumo MF-

473.111897(different from LvhD4)

gnl|WIGSC 573|kpneumo B







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].

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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.


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

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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.

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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.


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

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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)).


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.


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.

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