FEMS Microbiology Reviews, 39, 2015, 81–95
doi: 10.1111/1574-6976.12085
Review Article
REVIEW ARTICLE
Towards an integrated model of bacterial conjugation
Elena Cabezón, Jorge Ripoll-Rozada, Alejandro Peña, Fernando de la Cruz
and Ignacio Arechaga∗
Departamento de Biologı́a Molecular, Instituto de Biomedicina y Biotecnologı́a de Cantabria, IBBTEC,
(Universidad de Cantabria, CSIC) Santander, Spain
∗ Corresponding author: Ignacio Arechaga, Instituto de Biomedicina y Biotecnologı́a de Cantabria, PCTCAN, 39011 Santander, Spain,
Tel.: +(34) 942 202033; Fax: +(34) 942 201945; E-mail: [email protected]
One Sentence Summary: Bacterial conjugation is one of the main mechanisms for the dissemination of antibiotic resistance genes. Here, we suggest a
model that integrates the mechanism of conjugation with Type IV secretions systems biogenesis and substrate transport across the secretion channel
and we propose alternative approaches to use this knowledge in the fight against the spread of antibiotic resistance.
Present Address. Jorge Ripoll-Rozada, Instituto de Biologia Molecular e Celular, Porto, Portugal. Alejandro Peña, Cancer Research UK DNA Repair
Enzymes Group, Genome Damage and Stability Center, University of Sussex, Falmer, Brighton, UK
Editor: Christroph Dehio
ABSTRACT
Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the
dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a
membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in
bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific
inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights
recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport
by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This
model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating
expansion of antibiotic resistance.
Key words: secretion systems; bacterial conjugation; horizontal gene transfer; antibiotic resistance; pilus biogenesis;
macromolecular assemblies
INTRODUCTION
Over the last half a century, human health has profited from
the discovery and use of antibiotics to fight against infectious
diseases. However, after many years of extensive use, bacteria
fight back and our arsenal of antibiotics seem not to be enough
to cope with the avalanche of multi-resistant new pathogens
(McGowan and Tenover, 2004; Payne et al., 2007; Hawkey, 2008;
Boucher et al., 2009). Although new antibiotics are emerging in
the pipeline (Fischbach and Walsh, 2009; Gould and Bal, 2013),
very few have reached the market in the last four decades. One
of the main mechanisms whereby bugs become resistant to an-
tibiotics is the acquisition of antibiotic resistance genes by bacterial conjugation (Mazel and Davies, 1999; Waters, 1999; de la
Cruz and Davies, 2000). Therefore, the search for specific conjugation inhibitors is of special interest in the fight against these
superbugs. In this context, the recent development of new EcoEvo drugs has opened a promising strategy to control plasmid
dissemination using conjugation inhibitors (Baquero et al., 2011).
Conjugation is a process by which genetic material is transferred from a donor cell to a recipient cell. The transfer of
these conjugative genes requires a sophisticated machinery
that ensures DNA mobilization [brought about by MOB genes
Received: 28 March 2014; Accepted: 13 August 2014
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Figure 1. Genetic structure of the Mpf and Dtr regions of conjugative plasmid R388. Genes encoding proteins involved in mating pair formation (Mpf) are organized in
operons which are separated from those involved in DNA processing and transfer (Dtr). Mpf genes encode proteins that assemble in a large macromolecular structure
called Type IV secretion system, whereas Dtr genes encode proteins that bind to the DNA at the origin of transfer region, oriT, forming an structure called relaxosome.
This modular gene organization is shared by most conjugative systems, showing a high degree of gene synteny conservation. Agrobacterium tumefaciens nomenclature
for VirB/D proteins is indicated on top of each trw gene.
(Garcillan-Barcia et al., 2009; de la Cruz et al., 2010)] and
mating pair formation (organized by the MPF genes). These
genes can be encoded by an autonomous replicating plasmid (Smillie et al., 2010) or by integrative conjugative elements
(ICE) inserted in the chromosome (Wozniak and Waldor, 2010;
Guglielmini et al., 2011). Conjugation in Gram-negative bacteria is mediated by the Type IV secretion system (T4SS), a large
macromolecular complex involved in substrate transport and
pilus biogenesis. T4SSs are implicated not only in bacterial conjugation, but also in the secretion of virulence factors to eukaryotic cells. Many effectors secreted by T4SS are virulence factors
involved in pathogenic diseases, such as brucellosis, whopping
cough, cat scratch disease, pneumonia or gastric ulcer, caused
by bacterial infection with Brucella suis, Bordetella pertussis, Bartonella henselae, Legionella pneumophila or Helicobacter pylori, respectively (Vogel et al., 1998; Burns, 2003; Seubert et al., 2003;
Schulein et al., 2005; Nystedt et al., 2008; Engel et al., 2012;
Myeni et al., 2013). Excellent reviews on T4SSs have appeared
recently (Waksman and Fronzes, 2010; Christie et al., 2014;
Waksman and Orlova, 2014), and we are not going to dwell on
the general architecture of these systems. Instead, we will focus
on the issues that pertain specifically to conjugative systems,
analysing recent advances that have increased our understanding on the structure and function of conjugative T4SS.
Conjugative systems can be considered as a particular subfamily of T4SS. This subfamily is unique among bacterial secretion systems as it is not only able to transport protein effectors
but also DNA (covalently bound to a pilot protein) [for recent reviews see (de la Cruz et al., 2010; Zechner et al., 2012)]. DNA transfer during bacterial conjugation provides plasticity to bacterial
genomes, constituting one of the main mechanisms of horizontal gene transfer (Frank et al., 2005). Conjugative T4SSs are essential for mating pair formation (MPF) and, therefore, genes
encoding T4SS proteins are called MPF genes. Conjugative plasmids encode their own set of MPF genes and are, hence, selftransmissible. Other plasmids lack their own set of MPF genes
and depend on the T4SS of a conjugative plasmid to be mobilized. Accordingly, four distinct MPF families could be described
in Proteobacteria: MPFF (characterized by the conjugative plasmid F), MPFI (referring to IncI plasmids like RP4), MPFT , (based on
Agrobacterium tumefaciens Ti plasmid) and MPFG (related to ICEs)
(Smillie et al., 2010). Signatures of some MPF genes have also
been found in Cyanobacteria, Firmicutes, Bacteroides, Actinobacteria
and Archaea (Guglielmini et al., 2011), suggesting that conjugation is an ancient process that arose very early among ancient
Proteobacteria and then it spread out to all clades of prokaryotes
(Guglielmini et al., 2012).
MPFT systems encode the simplest T4SS, consisting of
11 proteins, named VirB1 to 11 after A. tumefaciens T4SS
(Christie et al., 2005). These 11 proteins, together with the
coupling protein VirD4, assemble into a macromolecular com-
plex that spans the inner and outer membrane and the
periplasm in between. The genes encoding these proteins are
organized in operons under the control of different promoters, with a clear distinction between those involved in mating pair formation (Mpf) and DNA transfer replication (Dtr)
(Fernández-López et al., 2006; de la Cruz et al., 2010). Figure 1
shows the organization of these genes in R388 plasmid, which
together with F and A. tumefaciens Ti plasmids are the best
characterized conjugative systems. This gene architecture is relatively well conserved in most conjugative systems (FernándezLópez et al., 2006; Guglielmini et al., 2012), which is a striking
feature considering that gene synteny is an evolutionary trait
that is quickly lost in evolution (Mushegian and Koonin, 1996;
Itoh et al., 1999). Synteny conservation is also observed in other
systems involved in the assembly of large multi-subunit complexes, such as the bacteriophage capsid (Brussow and Hendrix,
2002) or the archaeal (Patenge et al., 2001) and eubacterial flagella (Canals et al., 2006). Therefore, synteny conservation seems
to have an adaptive value, perhaps harnessing information related to the temporal programme of expression of the elements
of the complex.
T4SS architecture is relatively well preserved regardless
of its biological function (bacterial conjugation, secretion
of virulence factors into eukaryotic cells or DNA uptake)
(Waksman and Fronzes, 2010). Four protein domains can be distinguished in T4SS: the pilus, the core channel complex, the
inner membrane platform and the hexameric ATPases at the
base of the channel that supply the energy for pilus biogenesis and substrate transport (see Fig. 2 for subunit composition).
A near complete structure of T4SS from R388 plasmid has been
recently published (Low et al., 2014), revealing the general architecture of the secretion system (Fig. 2). This new structure has
provided new insights into T4SS assembly. However, some of the
components of the T4SS, such as the pilus or the VirB11 traffic
ATPase, are missing in the structure. Here, in the light of this new
structure and the genetic, biochemical and structural information provided by numerous laboratories along the last years, a
description of the architecture, biogenesis and function of the
four different T4SS domains is provided.
PILUS BIOGENESIS
A prevalent way for bacteria to accomplish adhesion to recipient cells is by the use of external appendages of different sizes
but similar helical structure known as pili (Bradley and Cohen,
1976; Achtman et al., 1978; Lai and Kado, 2000; Schroder and
Lanka, 2005; Thanassi et al., 2012). At the distal end of these filamentous structures, there is a molecule called adhesin (Aly and
Baron, 2007), which plays an essential role in cell-to-cell contact
(Anthony et al., 1994; Dehio, 2004). The major component of the
T4SS pilus is a hydrophobic protein named pilin or VirB2 (TrwL
Cabezón et al.
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tion appears to ensure the correct assembly of the F-pilus filament (Anthony et al., 1999).
Step 2. Pilin extraction from the inner membrane
Figure 2. Architecture of a conjugative T4SS. Four distinct domains can be distinguished in T4SS: the pilus, the core complex, the inner membrane platform
and the cytoplasmic ATPases. The pilus is formed by a helical assembly of pilin
(VirB2) molecules (red) with adhesin molecules (VirB5) at the distal end (dark
green). The core complex can be divided in two subcomplexes: the outer membrane cap, formed by VirB7, VirB9 and the C-terminus of VirB10, with a 14-fold
symmetry (depicted in deep blue colour); and the periplasmic domain (magenta),
formed by most of VirB8, VirB10 and the N-terminus of VirB6. The inner membrane platform (magenta), consisting of the transmembrane domains of VirB3,
VirB6, VirB8 and VirB10, is not well-defined in the EM structure by Low et al. (2014)
and, therefore, the location of these proteins cannot be precisely defined. Finally,
attached to the inner membrane are three hexameric ATPases: VirB4, VirB11 and
the coupling protein VirD4. In the EM structure, two hexameric barrels of VirB4
(yellow), anchored to the inner membrane through VirB3 (orange), were present,
whereas VirB11 (green) and VirD4 (salmon) were absent. The figure was created
using the EM structure comprising subunits VirB3 to VirB10 (Low et al., 2014)
(Electron Microscope Data Bank accession number: emd-2567), and the X-ray
structures of TrwB (VirD4) (Gomis-Ruth et al., 2001) (1e9r.pdb) and Brucella suis
VirB11 (Hare et al., 2006) (2gza.pdb), filtered at 2 nm resolution. For modelling
VirD4 transmembrane domains, the coordinates of Escherichia coli ATPase subunit c (Rastogi and Girvin, 1999) (1c17.pdb) were used.
in plasmid R388) (Fullner et al., 1996; Lai and Kado, 1998), being
the adhesin VirB5 (TrwJ in R388) a minor component (SchmidtEisenlohr et al., 1999). Although pilus biogenesis in T4SS is an
intricate process that is not well understood, a picture is emerging in which different steps leading to pilus formation can be
distinguished (Fig. 3).
Step1. Prepilin processing
Pilin proteins (VirB2) are synthesized as prepilin precursor
molecules, which contain an N-terminal leader signal sequence. Once in the inner membrane, the N-terminal leader sequence is processed by a signal peptidase I, presumably LepB
(Majdalani et al., 1996; Eisenbrandt et al., 2000; Lai et al., 2002). In
some VirB2 homologues, the N-terminal residue of the cleaved
protein binds covalently to the C-terminus, resulting in a mature, cyclic pilin (Eisenbrandt et al., 1999; Lai and Kado, 2002).
However, not all VirB2 homologues adopt this cyclic conformation. Some VirB2-homologues, such as TraA of plasmid F,
retain their linear sequence, probably by modification of the
N-terminus by N-acetylation (Moore et al., 1993). Pilin acetyla-
Because of their hydrophobic nature, pilin molecules can partition into the lipid bilayer without the additional help of any
other component of the T4SS (Paiva et al., 1992). Once the protein is accumulated in the inner membrane and, upon reception
of an unknown signal that triggers conjugation (Silverman, 1997;
Hwang and Gelvin, 2004), pilin molecules are dislocated from the
membrane towards the periplasmic space with the assistance of
VirB4/TrwK and VirB11/TrwD ATPases (Kerr and Christie, 2010).
VirB4 seems to act as the primary dislocation motor, whereas
VirB11 would act as a modulator of the VirB4 dislocase activity,
inducing conformational changes in the pilin.
VirB5 is essential for the incorporation of VirB2 to the
pilus (Schmidt-Eisenlohr et al., 1999; Lai et al., 2000). VirB5
stable expression is dependent on the presence of VirB6
(Hapfelmeier et al., 2000), a polytopic inner membrane component of T4SS. VirB5 localizes at the pilus distal end (Aly and
Baron, 2007) although it has also been found in the cytoplasm
and the inner membrane fractions of pili-producing bacteria
(Thorstenson et al., 1993). The crystal structure of TraC, the
VirB5 homologue in the conjugative plasmid pKM101, revealed
an elongated α-helical structure which, presumably, is involved
in cell-to-cell contact (Yeo et al., 2003; Hwang and Gelvin, 2004).
Interestingly, in contact independent T4SS, such as that of Bordetella pertussis, VirB5 is absent, but not a VirB2-like protein (PtlA)
(Weiss et al., 1993; Craig-Mylius and Weiss, 1999). As VirB2, VirB5
also possesses an N-terminal leader signal, likely to be processed by a signal peptidase I. However, in contrast to VirB2,
the mature VirB5 is not hydrophobic and, therefore, it is not expected to partition into the inner membrane. Upon cleavage of
VirB5 leader sequence, the mature protein would be exported to
the periplasm. This step is likely to trigger pilus assembly.
Steps 3 and 4. Pilus elongation
Packing geometry of VirB2 into the assembled pilus would be
in such a way that hydrophobic patches of the protein will
be buried at the interface between adjacent pilin monomers
(Marvin and Folkhard, 1986). These hydrophobic-driven interactions would promote the oligomerization of pilin molecules
into bundles which, in turn, would result in the formation of the
pilus, as observed in the structure of F-pili (Wang et al., 2009). The
mechanism driving conjugative pili assembly remains unclear,
and it is unknown whether pili assembly initiates from the inner membrane platform at the base of the T4SS or from the cap
complex at the outer membrane. Pilus polymerization from the
inner membrane might be driven by a mechanism similar to that
of T2SS and Type IV pilus assembly (reviewed in Filloux, 2004;
Craig and Li, 2008; Thanassi et al., 2012). This model of pilus
growth from an inner membrane base platform is supported by
the finding of VirB2 in both the inner and outer membranes in
cell fractionation experiments (Shirasu and Kado, 1993). Interestingly, in Type II secretion systems, the minor pseudopilins nucleate pilus assembly (Cisneros et al., 2012), reinforcing the idea
that VirB5 might also act by initiating filament assembly in T4SS.
However, this model of pilus elongation from the inner membrane would be incompatible with the recently solved structure
of T4SS from conjugative plasmid R388 (Low et al., 2014) in which
a central stalk formed by VirB6/VirB8 subunits rises from the inner membrane occupying the putative position of a pilus at this
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location (Fig. 2). On the basis of this structure, pilus biogenesis
might only occur in the periplasm, with the VirB8/VirB6 central
stalk acting as a nucleation platform.
Little is known about the size and morphology of VirB-like
conjugative pili. In contrast, the morphology of F-pili is well
characterized (Wang et al., 2009). They are flexible, long appendages (up to 20 μm), with an outside diameter of 8.5–9.5 nm
and a central lumen of 3 nm. The outer diameters of Type IV
(T4P), Type I and P pili are also around 6 to 9 nm wide
(Hahn et al., 2002; Mu and Bullitt, 2006; Craig and Li, 2008).
Therefore, VirB-like conjugative pili might have similar outer
dimensions. Considering the inner diameter of the T4SS outer
membrane ring, which is 2–3 nm, (Chandran et al., 2009;
Fronzes et al., 2009; Low et al., 2014), it is difficult to envisage
pilus polymerization within the secretion channel without a big
conformational change in the distal end of the core complex.
Therefore, an alternative scenario in which pilin polymerization
occurs at the outer membrane is also plausible. That is the case,
for instance, of the Type I pilus in the chaperone usher (CU) pathway (Allen et al., 2012), where the folding energy released by each
pilin subunit drives polymerization (Fig. 3).
In the Type IV pilus (T4P) apparatus, pilus extension and
retraction are mediated by PilB and PilT, two hexameric ATPases
located in the inner membrane (Merz et al., 2000; Mattick, 2002;
Craig and Li, 2008). In contrast to the T4P systems, conjugative
pilus retraction has been a puzzling issue for a long time.
Demonstration that conjugative pilus can retract came from
fluorescent experiments in which F-pilus retraction could
be visualized in real time (Clarke et al., 2008). However,
evidence for retraction of short-rigid pili like those of
Figure 3. T4SS pilus biogenesis. Step 1 - Pilin molecule precursors (VirB2), containing an N-terminal leader sequence, partition into the inner membrane where they
are processed by a signal peptidase, presumably LepB. Pilin molecules accumulate in the inner membrane until a specific signal triggers pilus biogenesis. Step 2 - Pilin
molecules are dislocated from the inner membrane into the periplasm by VirB4 with the assistance of VirB11. The peptidoglycan layer is digested by the transglycosylase
VirB1. In some systems, like in Bordetella pertussis, this transglycosylase activity is carried out by the VirB8 orthologue PtlE. Step 3 – VirB8 also acts as an assisting factor
for the assembly of the core complex. The channel is not yet fully assembled, and its inner diameter is wide enough to accommodate the emergent pilus. Pilus
elongation could occur by two different mechanisms: from the inner membrane platform, as T2SS and T4P, or from the outer membrane ring, as Type I pilus. Step
4 – Assembly of the core complex is completed, and the channel adopts its final conformation. Substrate secretion would only be possible if the central stalk (see
Fig. 2) is unplugged from the inner pathway of the secretion channel. Alternatively, the central stalk might act as a nucleation platform for pilus biogenesis, with pilus
polymerization leading to the ejection of the substrate (Note – For simplicity, only one of the VirB4 hexamers of the T4SS is shown).
Cabezón et al.
R388-like conjugative systems is still missing. It has been
suggested that F-pilus retraction does not require energy
(Frost et al., 1985). Instead, F-pilus assembly and retraction would be similar to the filamentous phage ‘telescopic
inovirus’ model (Marvin, 1989), whereby pilus rotation
along its longitudinal axis drives assembly and retraction
(Clarke et al., 2008).
ASSEMBLY OF THE SECRETION CHANNEL
(CORE COMPLEX)
In recent years, several advances have allowed to
gather a glimpse on the structural architecture of T4SS
(Chandran et al., 2009; Fronzes et al., 2009; Low et al., 2014),
providing information on the organization of the secretion
channel. This channel is formed by a large structure (1.05 MDa),
known as the core complex, which spans across the inner and
outer membranes and the periplasm in between. The structure
comprises 14 monomers of three distinct proteins (VirB7, VirB9
and VirB10). Below, some of their most salient properties are
described.
VirB7 is a small lipoprotein inserted in the outer membrane
(Fernandez et al., 1996a) firmly associated by disulphide bridges
with VirB9 (Anderson et al., 1996; Bayliss et al., 2007), which is
mainly involved in maintaining the integrity of the core complex (Fernandez et al., 1996b). VirB7 contains a globular domain
that shares structural similarities with the N0 domains of Type
IV pilus PilQ, T2SS GspD, T3SS EscC and filamentous phage pIV
secretins (Souza et al., 2011).
VirB9 is a hydrophilic protein also located at the outer membrane. The C-terminal domain of VirB9 contains a β-sandwich
fold around which VirB7 binds, forming a stable outer membrane channel complex (Bayliss et al., 2007). VirB9 interacts
with VirB10 and, hence, contributes to the stabilization of
the secretion channel (Beaupre et al., 1997). The crystal structure of this outer membrane complex formed by VirB7, VirB9
and the C-terminal end of VirB10 has recently been solved
(Chandran et al., 2009), revealing a cap structure with an inner
pore of 3.2 nm.
VirB8 is another component traditionally associated with the
channel complex (Das and Xie, 2000; Kumar et al., 2000). VirB8
is anchored to the inner membrane by its N-terminal region
(Thorstenson and Zambryski, 1994; Buhrdorf et al., 2003). The
three-dimensional structure of a periplasmic fragment (residues
77-239) of the VirB8 protein of B. suis consists of a large extended β-sheet and five α-helices, with an overall globular fold
(Terradot et al., 2005). VirB8 interacts with VirB9 and VirB10
(Das and Xie, 2000) which suggests that it plays an essential
role in the assembly of the secretion channel (Kumar et al., 2000;
Judd et al., 2005). In the recent EM structure of the T4SS from
conjugative plasmid R388 (Low et al., 2014), VirB8 appears to be
associated with VirB6 forming a central stalk structure that occludes the pore of the secretion channel (Fig. 2). This disposition
of the central stalk formed by VirB8/VirB6 does not seem to be
compatible with a channel able to secrete any type of protein
or DNA substrate. It is possible that this structure represents an
intermediate step during T4SS and pilus assembly in which the
central stalk plays an essential role. Then, upon completion of
T4SS biogenesis, this central stalk could be unplugged from the
channel pore, allowing the passage of substrates. This notion is
supported by the fact that VirB8 seems to be loosely attached to
VirB9 and VirB10, as reflected by its absence from the EM structure of the core complex, despite of being initially co-expressed
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(Fronzes et al., 2009). In summary, VirB8 could be an assembly
factor, needed for complex assembly but not a component of the
secretion channel.
VirB10 is the largest protein in the core complex, spanning
the inner and outer membranes and the periplasmic space
(Jakubowski et al., 2009). VirB10 shares structural similarities
with TonB-like proteins (Cascales and Christie, 2004a), such as
a common bitopic membrane topology and a prolin-rich extended region in the periplasm (Evans et al., 1986). These structural features were later confirmed by the crystal structure of
the periplasmic domain (residues 146-376) of ComB, the VirB10
homologue in H. pylori (Terradot et al., 2005). In this crystal
structure, the VirB10 periplasmic domain was shown to form
dimers, confirming data obtained by two-hybrid experiments
(Ding et al., 2002). Therefore, given that there are 14 copies of
VirB10 in the core structure (Fronzes et al., 2009), it could be assumed that there is a heptameric repetition of the dimeric unit
observed in the crystal structure of ComB periplasmic domain.
However, the subunit packing observed in this structure, with
the N-termini of each monomer at opposite ends, is not compatible with that observed in the core complex structure.
One of the most interesting features of the EM structures of
the T4SS (Fronzes et al., 2009; Low et al., 2014) is the size of the
inner channel cap constriction at the outer membrane domain
of the core complex (10–20 Å). Such a small size implies that
any protein substrate crossing the channel should be in an unfolded state, unless a large aperture of the channel takes place.
Interestingly, the crystal structure of the outer membrane part
of the core complex, formed by 14 copies of VirB7, VirB9, and the
C-terminal domain of VirB10 (Chandran et al., 2009) revealed a
much larger dimension of the inner ring (32 Å). Therefore, the EM
and the crystal structures significantly differ in the inner diameter dimensions of the pore. Comparison of the outer membrane
cap crystal structure (Chandran et al., 2009) with that of the EM
core complex (Fronzes et al., 2009) revealed that the outer transmembrane helices of VirB10 in the crystal structure were in a
‘relaxed’ conformation, forming a 45◦ angle relative to the vertical disposition observed in the EM structure. This conformational change could be due to the fact that the cap crystal structure was obtained after removal by proteolysis of the N-terminal
half of VirB10. Interestingly, comparison between the EM structures of the pKM101 core complex (Fronzes et al., 2009) and R388
(Low et al., 2014) revealed a flexibility of the VirB10 N-terminus,
which fluctuates from a compacted conformation under the I
layer of the core complex and an extended conformation in the
R388 structure. These conformational differences could reflect
natural occurring transitions but more effort is needed to understand the arrangement of the VirB10 N-terminus within the
core complex and its interactions with the stalk domain formed
by VirB8/VirB6 and, probably, VirB3. In any case, it seems that
VirB10 N-terminus plays an essential role in the transmission of
the signal from the inner membrane to the rest of the core complex, which could result in the opening of the secretion channel.
ATP-dependent conformational transitions have been
reported for VirB10 (Cascales and Christie, 2004). Protease
susceptibility experiments showed that VirB10 can adopt two
different conformations: ATP-independent and ATP-dependent
‘energized’ forms, although only the energized conformation
is able to form a complex with VirB9. Such structural transitions occur in response to ATP utilization by the VirD4 and
VirB11 ATPases (Cascales and Christie, 2004). Interactions of
VirB10 with the coupling protein VirD4 have been reported
(Gilmour et al., 2003; Llosa et al., 2003; Cascales et al., 2013).
These interactions take place in the inner membrane (de
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Paz et al., 2010), which suggests that conformational changes
at the base of the channel are transmitted across the periplasmic and OM domains of VirB10, leading to the aperture of
the external cap. Interestingly, heteromeric transmembrane
interactions in VirB10 have been found to be essential for
T-pilus biogenesis but not for substrate secretion (Garza and
Christie, 2013).
VirB10 is also able to interact with VirB4. A low-resolution EM
structure of the core complex bound to the VirB4-homologue of
the conjugative plasmid pKM101 revealed an intimate relation
between VirB4 and the channel complex (Wallden et al., 2012).
However, the recently solved structure of the T4SS from R388
shows that the VirB4 N-terminus is not embedded in the I
layer of the core complex. Instead, an association to the complex through the integral membrane protein VirB3 seems a
more plausible explanation. This idea is supported by the
finding of VirB3–VirB4 fusion proteins in the IncX branch
(Batchelor et al., 2004). It is enticing to speculate that the ATPase
activity by VirB4 induces conformational changes in the core
complex during pilus biogenesis whereas VirD4 does it during
substrate transport.
MOLECULAR MOTORS THAT DRIVE PILUS
SYNTHESIS AND SUBSTRATE TRANSFER
Type IV secretion assembly and substrate transport are processes that require energy, usually coming from ATP hydrolysis
catalysed by specific ATPases (Peña and Arechaga, 2013). VirB4
and VirB11, two ATPases encoded by T4SS (Rivas et al., 1997;
Arechaga et al., 2008), have been suggested to be involved in
pilus biogenesis and substrate transport (Rabel et al., 2003).
In addition, conjugative systems have a third ATPase, VirD4
(Tato et al., 2005), termed coupling protein (T4CP) as it couples relaxosome DNA processing to the transport of the nucleoprotein
complex across the T4SS (Cabezón et al., 1997; Llosa et al., 2002).
VirB4
VirB4 is the only ATPase that is present in all T4SS and, therefore, the presence of a VirB4-like protein constitutes a signature
of a T4SS (Alvarez-Martinez and Christie, 2009). For instance,
in F-plasmids, VirB4-like TraC is the only ATPase required for
pilus production (Lawley et al., 2003). VirB4 proteins are also the
largest and most conserved constituents of T4SS (FernándezLópez et al., 2006; Guglielmini et al., 2012). The protein consists
of two distinct domains: an N-ter (NTD) and a C-terminal domain (CTD). The CTD of VirB4 proteins is ancestrally related
to the Type IV coupling proteins T4CP (Guglielmini et al., 2012)
and contains the Walker A and Walker B motifs, responsible for the ATPase activity (Arechaga et al., 2008). The crystallographic structure of the CTD of a VirB4 homologue in
Thermoanaerobacter pseudethanolicus has recently been reported
(Wallden et al., 2012). The structure turned out to be strikingly
similar to the computer generated models of the CTD of A. tumefaciens (Middleton et al., 2005) and conjugative R388 VirB4 homologues (Peña et al., 2011), which were built using the structure
of TrwB (Gomis-Ruth et al., 2001), the VirD4 homologue in plasmid R388 (see Fig. 1), as a template. VirB4CTD also shares a high
degree of structural similarity with the CTD of FtsK and the soluble domain of TrwB (Cabezón et al., 2012). FtsK and TrwB are
DNA pumps involved in bacterial chromosome segregation and
bacterial conjugation, respectively. These three motor proteins
seem to have evolved from a common ancestor (Iyer et al., 2004;
Guglielmini et al., 2012) and, interestingly, a DNA-binding ac-
tivity has been recently demonstrated in different VirB4 homologues (Li et al., 2012; Peña et al., 2012). At the C-terminal end
of VirB4 proteins, there is a conserved α-helical region, which
is absent in TrwB. This α-helical region is a regulatory domain
that down-regulates the ATPase activity of the protein, probably
to prevent a futile waste of energy in the absence of a biological activity, with a mechanism that resembles the regulation of
other ATPases (Peña et al., 2011).
In contrast to the CTD, there is little information about
the NTD of VirB4 proteins. The IncX subfamily of VirB4 proteins is characterized by the presence at the N-terminus of an
extra region corresponding to a fusion with a VirB3 protein
(Batchelor et al., 2004). VirB3 proteins are polytopic inner membrane proteins that require VirB4, VirB7 and VirB8 for stabilization (Mossey et al., 2010). VirB3 has also been found to
co-elute with VirB2 and VirB5 in membrane fractionation experiments (Yuan et al., 2005), suggesting that VirB3 is essential in
pilus biogenesis. VirB4 has been reported to be an integral cytoplasmic membrane protein (Dang and Christie, 1997). However, computer predictions of VirB4 topology are negative for
transmembrane spans in most of the VirB4 members, excluding
those of the IncX branch, which contain a VirB3-like sequence at
the N-termini (Arechaga et al., 2008). Moreover, TraB, the VirB4
homologue in the conjugative plasmid pKM101, has been isolated both, as a soluble and as a membrane-associated form
(Durand et al., 2010). It is altogether very likely that VirB3 acts
as an anchor of VirB4 to the membrane, assisting it in its VirB2
dislocase function (Kerr and Christie, 2010).
Although there is not any high-resolution structure of the
VirB4NTD available, a low-resolution structure of the full-length
VirB4 homologue of the conjugative plasmid R388 has been
reported (Peña et al., 2012). This structure was obtained in its
hexameric form, which is likely to be the catalytic active conformation of the enzyme (Arechaga et al., 2008). The oligomeric
state of VirB4 proteins has been largely under dispute. Initial reports on the oligomeric state of A. tumefaciens VirB4
stated that it assembled minimally as a dimer, although higherorder structures were not discarded (Dang et al., 1999). On the
other hand, the VirB4 homologue in the conjugative plasmid
pKM101 has been reported to be present both as dimer and hexamer, being the hexameric form soluble and catalytically active
and the dimeric form inactive and membrane-associated (Durand et al., 2010). This debate on the oligomeric state of VirB4
seems to have been resolved by the recent structure of the T4SS
from R388 (Low et al., 2014), which clearly shows that VirB4
forms hexamers at the base of the secretion channel, as previously suggested (Middleton et al., 2005; Arechaga et al., 2008;
Peña et al., 2012). Moreover, this structure shows not only one
but two hexamers attached to the inner membrane domain of
the T4SS. The implications of this arrangement in the molecular mechanism are however still unclear.
VirB11
VirB11 proteins belong to a large family of hexameric AAA+ traffic ATPases, which includes proteins involved in Type II secretion
and in Type IV pilus and flagellar biogenesis (Planet et al., 2001).
VirB11 proteins consist of two domains, an N-ter (NTD) and a Cter (CTD), connected by a flexible linker of variable length. The
VirB11NTD has been suggested to interact with the membrane
(Yeo et al., 2000), whereas the VirB11CTD contains the Walker
A and Walker B motifs and is thus involved in ATP hydrolysis
(Rivas et al., 1997; Ripoll-Rozada et al., 2012). The linker region between both domains has been proposed to play a key role in en-
Cabezón et al.
zyme catalysis (Hare et al., 2006). In fact, although the crystallographic structures of two non-conjugative homologues, HP0525
from Helicobacter pylori (Yeo et al., 2000; Savvides et al., 2003)
and VirB11 from Brucella suis (Hare et al., 2006), revealed similar hexameric rings, the dynamics of hexamer formation and
the monomer-monomer interactions are completely different.
These differences are produced by a large domain swap of the
central linker, which is much larger in the Brucella homologue
than in the Helicobacter counterpart (Hare et al., 2006). Based on
biochemical and structural evidence, a common model for the
mechanism of action of these secretion ATPases has been proposed (Yamagata and Tainer, 2007; Ripoll-Rozada et al., 2012).
According to this model, the VirB11NTD of the nucleotide-free
form of the enzyme would be pivoting over the flexible linker.
Binding of magnesium and ATP would lock the enzyme in
a closed conformation. Upon ATP hydrolysis, a Mg-ADP state
would be stabilized and, consequently, at physiological Mg2+
concentrations this Mg-ADP state would remain in an inhibited, closed conformation. A specific signal (for instance, substrate binding or release) would unlock this state, resuming
the catalytical cycle by releasing the ADP for the next turnover
(Ripoll-Rozada et al., 2012).
The biological function of VirB11 is still unknown. VirB11
seems to participate in pilus biogenesis by assisting VirB4 dislocase activity on VirB2 (Kerr and Christie, 2010). Actually,
interactions between VirB11 and VirB4 have been observed
(Draper et al., 2006; Ripoll-Rozada et al., 2013). These interactions
depend on the length of the linker region, as inferred from experiments in which VirB4 was tested for interactions with VirB11
homologues of different linker size (Ripoll-Rozada et al., 2013).
These interactions seem to be unstable as judged by the
fruitless attempts to purify stable VirB4–VirB11 complexes
(J. Ripoll-Rozada, unpublished) and the absence of VirB11 in the
T4SS structure (Low et al., 2014).
VirB11 has also been suggested to play an essential
role in the first steps of the DNA translocation pathway
(Atmakuri et al., 2004b), as shown by transfer DNA immunoprecipitation assays (Cascales and Christie, 2004b). Supporting
this dual functionality, mutations in VirB11 uncouple T-pilus
production from substrate translocation (Sagulenko et al., 2001).
It has been suggested that VirB11 acts as a molecular switch
between pilus biogenesis and substrate transport by replacing
VirB4 and VirD4 ATPases at the entrance of the secretion channel
(Ripoll-Rozada et al., 2013). It is worth noting that VirB11, although widely distributed among conjugative systems, is not
found in all T4SS. MPFF plasmids, for instance, do not code for a
cognate VirB11 homologue (Frost et al., 1994; Lawley et al., 2003),
suggesting that there are important differences between MPFT
and MPFF conjugative systems, mainly in the morphology,
length and flexibility of the pilus. In DNA uptake T4SS, like H. pylori ComB system, a cognate VirB11 orthologue is also missing
(Karnholz et al., 2006). Therefore, in these systems, the function
of VirB11 seems to be dispensable or replaceable by other proteins with similar structure.
VirD4
The T4SS coupling protein (T4CP) or VirD4 in A. tumefaciens
is essential for substrate translocation but, in contrast to
VirB11 and VirB4 ATPases, is dispensable for pilus biogenesis
(Lai et al., 2000). The main function of conjugative coupling proteins is to drive the nucleoprotein susbtrate to the secretion
channel (Cabezón et al., 1997). The coupling function of T4CP
is mediated, on one side, by interactions with the relaxosome
87
auxiliary proteins (Disque-Kochem and Dreiseikelmann, 1997;
Tato et al., 2007) and, on the other side, by interactions with
the inner membrane domain of the core complex VirB10 protein
(Gilmour et al., 2003; Llosa et al., 2003; de Paz et al., 2010).
TrwB is a DNA-dependent ATPase (Tato et al., 2005). The
structure of the cytoplasmic, soluble domain of TrwB revealed
a hexamer with a six-fold symmetry and a central channel of
about 20 Å in diameter (Gomis-Ruth et al., 2001). The protein
presents structural similarity to hexameric molecular motors
such as F1 -ATPase, FtsK or ring helicases (Iyer et al., 2004), suggesting that TrwB also operates as a motor, using energy released
from ATP hydrolysis to pump ssDNA through its central channel
(Cabezon and de la Cruz, 2006; Gomis-Ruth and Coll, 2006). At
the N-terminal domain of TrwB, there are two transmembrane
α-helices which anchor the protein to the inner membrane. This
transmembrane domain also regulates nucleotide binding affinity (Vecino et al., 2010), and it is important in stabilizing the structure of the protein (Vecino et al., 2011).
TrwB ATPase activity is enhanced by TrwA (Tato et al., 2007),
an accessory protein, which together with the DNA and TrwC
forms the relaxosome of R388 plasmid. TrwA binds specifically
at the origin of transfer of the plasmid (oriT) as a tetramer
and facilitates TrwB assembly on DNA (Moncalian and de la
Cruz, 2004). The C-terminal domain of TrwA interacts with
TrwB, whereas its N-terminal domain is involved in DNA binding. In IncF plasmids, interactions between the coupling protein TraD and the accessory protein TraM have also been
reported (Lu and Frost, 2005) and the structure of the C-terminal
tail of TraD bound to the TraM tetramerization domain has
been solved (Lu et al., 2008). Intriguingly, the TrwA–TrwB system resembles the mechanism used by other DNA-dependent
molecular motors such as the RuvA-RuvB recombination system
(Yamada et al., 2002; Tato et al., 2007). Recently, it has been shown
that TrwB is a structure-specific DNA-binding protein, with very
high affinity for G4 DNA structures (Matilla et al., 2010). The
protein might be involved in resolving G4 secondary structures
that arise during conjugative DNA processing. Alternatively, this
higher affinity for G-quadruplex might reflect the need of a secondary DNA structure as a loading site for the motor. Further
experiments are needed to clarify the in vivo function of Gquadruplex structures in the conjugative process.
SUBSTRATE TRANSPORT
Biochemical and genetic analysis of T4SS components provide
evidence supporting a model in which DNA/protein transfer and
pilus production are uncoupled and independent processes. A
pathway of interactions between the T-DNA substrate and the
different T4SS subunits during DNA transfer has been proposed
(Cascales and Christie, 2004b). However, despite the mounting
evidence describing the interactions between T4SS components
and the interactions between them and the translocated substrate, there are still a number of questions that remain open.
For instance, the involvement of the core complex subunits during substrate transport, the specific role of the three ATPases
or the fate of the pilus after the formation of the mating pair
are still not fully understood processes. Last but not least, an
important question that remains unanswered is the conformational state, native or unfolded, of the protein substrate during
translocation. Here, based on the information available up to
date, a detailed description of the events that take place during
DNA processing and transport in bacterial conjugation is provided (Fig. 4).
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FEMS Microbiology Reviews, 2015, Vol. 39, No. 1
Figure 4. Nucleoprotein transport by T4SS. Step 1 – Donor cells contact recipient cells. The cell-to-cell contact, mediated by the pilus, could be triggered by specific
factors in the recipient. At this stage, the auxiliary factors TrwA and IHF bind to the origin of transfer region (oriT) of plasmidic DNA and the relaxase cleaves it at the
nic site. Step 2 – Upon cell contact, the retraction of the extracellular pilus facilitates the interaction between membranes of donor and recipient cells, resulting in a
membrane fusion process. Simultaneously, or prior to this membrane fusion, the coupling protein drives the relaxosome towards the secretion channel. Step 3 – The
nucleoprotein substrate is transferred to TrwD/VrB11. The relaxase is unfolded and translocated through the channel covalently bound to the DNA. Step 4 – TrwK/VirB4
is displaced at the base of the secretion channel by the coupling protein, which assists DNA translocation in a 5 –3 direction.
Step 1. DNA recognition and relaxase cleavage reaction
Conjugative and mobilizable plasmids contain a cognate DNA
sequence, oriT, which is recognized by specific auxiliary factors
within the relaxosome. The relaxosome, called like this by analogy with the nucleosome, is formed by the relaxase and auxiliary
transfer proteins bound to a fragment of DNA several hundred
base pairs in length, which contains the origin of transfer (oriT)
(Fürste et al., 1989).
Relaxases are specific phosphodiesterases which cleave
DNA at a nic site within the oriT (Byrd and Matson, 1997;
Chandler et al., 2013). Relaxases are usually, but not always,
large multi-domain proteins (for a recent review see GarcillanBarcia et al., 2009). In all cases, the relaxase domain, which is
approximately 300 aa long, is located at the N-terminus of the
protein. At the C-terminus, a DNA helicase or a DNA primase
can be found. Occasionally, an extra domain of unknown function is also observed. For instance, TraI, the relaxase of plasmid
F, is a 182 kDa protein (Abdel-Monem et al., 1983) with three
distinct domains (Cheng et al., 2011): the relaxase (1–306 aa),
the helicase (309–1504) (Byrd et al., 2002) and a third domain,
comprising the last 252 aa, which is involved in DNA binding
(Guogas et al., 2009). In most cases, relaxases contain a conspicuous signature consisting of a histidine triad (3H motif)
Cabezón et al.
(Garcillan-Barcia et al., 2009), which is used by the protein to
bind divalent cations. This metal ion is essential for the relaxase
cleavage reaction (Boer et al., 2006) but not for DNA binding
(Lucas et al., 2010). An analogous histidine motif is also found
in nucleases involved in rolling circle DNA replication (RCR)
(Koonin and Ilyina, 1993; Chandler et al., 2013), suggesting that
the generation of a single-stranded DNA copy of the plasmid occurs by a similar RCR mechanism.
Crystal structures of the relaxase domains of TrwC of plasmid R388 (Guasch et al., 2003; Boer et al., 2006), TraI of plasmid F
(Datta et al., 2003; Larkin et al., 2005), MobA (Monzingo et al., 2007)
and the relaxase of Salmonella typhimurium pCU1 plasmid
(Nash et al., 2011) have been determined, providing details of the
cleavage reaction and ssDNA-protein interactions. The cleavage
reaction occurs via a nucleophilic attack by the hydroxyl group of
the relaxase catalytic tyrosine residue on the 5 -side of the DNA
phosphate. This transesterification reaction results in a covalent
linkage between protein and DNA (Guasch et al., 2003; GonzalezPerez et al., 2007). After the cleavage reaction, donor DNA synthesis begins from the 3 -end of the cleaved strand. Then, the
helicase domain displaces the old DNA strand, which is replaced
by the new synthesized DNA strand, and the covalently bound
nucleoprotein is transferred to the recipient cell.
The auxiliary proteins of the relaxosome are involved in the
regulation of gene expression. For instance, TraY and TraM in
IncF plasmids bind to the F tra operon promoters, which results
either in a positive or negative regulation of gene expression
(Schwab et al., 1993; Penfold et al., 1996; Silverman and Sholl,
1996; Taki et al., 1998). TraY and its orthologues, such as TrwA
in R388, are structurally related to DNA-binding proteins with
ribbon-helix-helix (RHH) motifs. Proteins with this RHH motif interact in antiparallel fashion with the major groove of the DNA
(Bowie and Sauer, 1990). An additional auxiliary factor IHF (integration host factor) binds to the DNA inducing a bending in
the oriT region (Rice et al., 1996; Seong et al., 2002) that helps
to bring TraY to the nic site. The auxiliary proteins are not absolutely required for linear DNA cleavage by MOBF relaxases,
but they seem to be essential for nicking activity on supercoiled
DNA (Garcillan-Barcia et al., 2009). The role of the different auxiliary proteins and the mechanism of cleavage and processing of conjugative DNA is described in further detail in (de la
Cruz et al., 2010).
Step 2. DNA driving to the membrane
Once the cleavage reaction has ended, the nucleoprotein complex must be recruited to the membrane channel to initiate
transfer (Llosa et al., 2002). This step is mediated by the coupling protein (Cabezón et al., 1997), which binds to the relaxosome with the assistance of the auxiliary proteins. Binding
of the coupling protein to the DNA is sequence unspecific but,
thanks to the auxiliary proteins that recognize a specific sequence in the oriT (Moncalian and de la Cruz, 2004), the coupling
protein is able to bind to an specific region within the relaxosome. As mentioned above, TrwB has been found to bind G4quadruplex DNA with much higher affinity than any other type
of DNA (Matilla et al., 2010). Therefore, it is possible that DNA
G-quadruplex structures can also act as loading sites for TrwB.
89
TrwC has been shown to display site-specific and recombinase
activities in the recipient cell (Draper et al., 2005). However,
the mechanism of transport across the T4SS channel is unknown. Given the large size of most relaxases (over 100 kDa),
it is unlikely that they can go through the T4SS channel in
a native state conformation and an unfolding mechanism
should be envisaged. Nothing is known about this unfolding
mechanism but it would involve the assistance of some kind of
unfoldase. In this regard, it is worth noting the proposed role
of the secretion ATPase superfamily proteins as traffic ATPases
(Planet et al., 2001). The overall structure of the traffic ATPase
VirB11 (Yeo et al., 2000; Hare et al., 2006) is highly conserved
among distantly related ATPases associated to other transport
systems (Peña and Arechaga, 2013). T3SS also have an AAA+
hexameric ATPase, InvC, which is a chaperone involved in the
release of the unfolded substrate to the secretion channel in
a process coupled to ATP hydrolysis (Akeda and Galan, 2005).
Interestingly, and similarly to TrwD (Rivas et al., 1997), InvC
fractionates both as cytoplasmic and as a peripheral inner
membrane protein. Therefore, a tempting hypothesis is that
TrwD/VirB11 binds and unfolds the relaxase in a similar manner
to that of InvC and then it delivers the unfolded substrate to
the inner membrane platform at the base of the T4SS secretion
channel. However, experimental evidence of interactions between VirB11 and the relaxase is still missing. The putative role
of VirB11 in substrate transport as an acceptor of the nucleoprotein complex from the coupling protein is supported by several
lines of evidence, such as the T-DNA immunoprecipitation (TrIP)
assays carried out in A. tumefaciens (Cascales and Christie, 2004).
Step 3b. Relaxase shooting
Once the nucleoprotein complex has been recruited to the T4SS
by the coupling protein and transferred to VirB11, the substrate
is delivered to VirB6 and VirB8 proteins in a process mediated
by VirB4 (Cascales and Christie, 2004). However, the mechanism by which the relaxase is transported across the secretion
channel is still unclear. In the VirB system of Brucella, this type
of signal sequence has been identified in several effectors (de
Jong et al., 2008; de Barsy et al., 2011; Marchesini et al., 2011).
This signal has been localized in some cases at the N-termini,
whereas in other cases this signal was located at the C-termini
of the translocated protein (de Jong et al., 2008). In contrast, relaxase translocation signals (TSs) in conjugative T4SS have been
localized to internal positions. The study on the relaxase TraI
of R1 and F plasmids (Lang et al., 2010), identified two internal
TSs (TSA and TSB), with conserved residues that defined a consensus sequence (G[E/D]R[L/M]R[V/F]T). The three-dimensional
structure of TSA has recently been reported (Redzej et al., 2013).
Based on conservation of the consensus sequence within a
RecD2-like domain of the proteins, an extension of these results
to other relaxases was proposed (Lang et al., 2010). Accordingly,
two TSs (TS1 and TS2) in the relaxase TrwC of plasmid R388
were also identified (Alperi et al., 2013). Mutations within TS1 region almost abolished conjugation by the T4SS of plasmid R388,
whereas mutations in both TSs barely altered DNA transfer by
the T4SS of B. henselae. These results seem to indicate that a single substrate can be recruited by two different T4SSs through
different signals (Alperi et al., 2013).
Step 3a. Relaxase unfolding
T4SS-dependent
transfer
of
relaxases
to
human
(Schulein et al., 2005), plant (Vergunst et al., 2000) and bacterial (Draper et al., 2005) recipient cells has been documented.
Step 4. DNA passage across the secretion channel
According to a shoot and pump model (Llosa et al., 2002), once
the relaxase, covalently bound to the DNA, is shot through the
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FEMS Microbiology Reviews, 2015, Vol. 39, No. 1
channel, TrwB wraps on the DNA as a hexamer and uses energy
derived from ATP hydrolysis to pump the DNA. The proposed
mechanism (Cabezon and de la Cruz, 2006) is a modified version
of the so-called binding change mechanism proposed for the F1 ATPase, which involves a sequential binding and hydrolysis of
ATP (Boyer, 1997). After the transfer of a complete copy of a single
DNA strand into the recipient cell, the relaxase would recognize
the nic site and it would carry out a second transesterification
reaction, resulting in re-circularization of the transferred strand
in the recipient (Draper et al., 2005).
One important question that remains unanswered is the extent of the conformational changes that take place in the core
complex channel. It is evident that these structural conformational changes should be large enough to permit the nucleoprotein passage across the T4SS. These dramatic conformational
transitions are likely to require energy, which might be provided by one of the two ATPases at the entrance of the secretion channel. Moreover, by protease susceptibility assays, it has
been shown that VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATPases. Such
an energy-induced conformational change in VirB10 is required
to form the complex with the outer membrane-associated proteins VirB9 and VirB7 (Cascales and Christie, 2004a). The mechanism resembles that of the TonB-dependent transporters, where
TonB also acts as an energy sensor (Postle and Kadner, 2003).
VirB10 shares structural similarities with TonB-like proteins
(Cascales and Christie, 2004a). TonB is also sequence and structurally related to the T2SS protein GspB. TonB and GspB act as
energy sensors of the proton motive force (pmf), and in some
cases, like in Klebsiella oxytoca, this is the only energy source
required (Possot et al., 1997). However, in other T2SS, ATP hydrolysis is required in addition to the pmf (Letellier et al., 1997).
The pmf has also been postulated to be the only energy source
required for substrate secretion by T3SS (Wilharm et al., 2004;
Paul et al., 2008), although ATP hydrolysis is essential for substrate unfolding (Akeda and Galan, 2005). Hence, the translocation of substrates across the T4SS might also require both the
pmf and ATP hydrolysis as energy sources.
Regardless of the source of energy, it has become evident
that VirD4 interacts with VirB10 during substrate transport
(Cascales et al., 2013). The interactions between these two
proteins would only be possible if the resident VirB4 protein, involved in pilus biogenesis, is displaced from the base
of the core channel. It has been suggested that VirB4 and
VirD4 could be interchanged on this location in the conjugative systems (Peña et al., 2012) in a process mediated by VirB11
(Ripoll-Rozada et al., 2013). Interactions between VirB4 and VirD4
have been reported (Li et al., 2012; Peña et al., 2012). Moreover, it
has been reported that VirB4/TrwK is able to inhibit the DNAdependent ATPase activity by TrwB (Peña et al., 2012), further
supporting the hypothesis of the formation of transient TrwB–
TrwK heterocomplexes at the base of the secretion channel.
in the cell (Garcillan-Barcia and de la Cruz, 2008). Little is known
about specific receptors in the recipient cell that allow pilus attachment. Interactions between the pilus tip and lipopolysaccharides (LPS) in the recipient cells have been reported
(Anthony et al., 1994; Gyohda et al., 2004), and modifications of
the cell wall LPS were found to affect efficient mating ability
(Watanabe et al., 1970). However, recent data have shown that
mutations in the LPS pathway had little effect on conjugation
and that no nonessential recipient E. coli genes played an essential role in conjugation. These results suggest that conjugation
would take place with little regard to the recipient cell constitution (Perez-Mendoza and de la Cruz, 2009).
Another important question that remains unsolved is the exact role of pili in DNA transfer. Although the most universally
accepted idea is that pili only provide cell-to-cell contacts, the
possibility that ssDNA travels through the lumen of the pili is
still debatable. F-pilus, for instance, presents a 20 Å central lumen (Marvin and Folkhard, 1986), which might well accommodate ssDNA. However, it is difficult to envisage the passage of
the protein substrate through the pilus. It could be also possible, especially in the short-rigid pili encoded by IncP and IncW
plasmids, that after engagement of the recipient cell, pili depolymerize facilitating the membrane contacts between donor and
recipient cells, leading to membrane fusion. At that stage, T4SS
could easily deliver its substrates in the interior of the recipient cell. This membrane fusion hypothesis would also explain
the mechanism of interaction between the entry exclusion factor in the recipient cell and T4SS components in the donor cell
(Garcillan-Barcia and de la Cruz, 2008). Alternatively, a mechanism similar to the T3SS injectisome, in which the pilus works
as a needle, shooting up the virulence factors directly into the
recipient cell, is also feasible (Galan and Wolf-Watz, 2006).
In summary, there are still challenging questions to be solved
to understand the intimate mechanisms of DNA and protein
transport in bacterial conjugation. Understanding these mechanisms is essential to develop strategies in the battle against
the dissemination of antibiotic resistance genes. Additionally,
a better understanding of the conjugative machinery could be
helpful in the development of farfetched biotechnological applications, such as gene therapy (Llosa and de la Cruz, 2005;
Schroder et al., 2011; Llosa et al., 2012) or plant transformation
(Tzfira and Citovsky, 2006).
FUTURE WORK
REFERENCES
Although much progress has been made in describing the
molecular architecture of the T4SS, these efforts have not been
accompanied by a better understanding of the dynamics of the
secretion mechanism. In DNA transfer systems, many essential
questions remain unsolved, such as the mechanism by which
the nucleoprotein complex enters in the recipient cell, overcoming the membrane barrier, or the mechanism by which entry exclusion proteins prevents the entry of a second identical plasmid
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ACKNOWLEDGEMENTS
This work was supported by the Spanish Ministerio de Economı́a
y Competitividad (MINECO) grants BFU2011-22874 (to E. C.
and I. A.) and BFU2011-26608, EU VII Framework Program
projects 282004/FP7-HEALTH-2011-2.3.1-2 and 612146/ICT-201310 (to FdlC).
Conflict of interest statement. None declared.
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