Molecular Microbiology (2002) 45(1), 1–8
MicroReview
Bacterial conjugation: a two-step mechanism for
DNA transport
Matxalen Llosa,1* F. Xavier Gomis-Rüth,2
Miquel Coll2 and Fernando de la Cruz1*
1
Departamento de Biología Molecular (Unidad Asociada
al CIB-CSIC), Universidad de Cantabria, C. Herrera
Oria s/n, 39011 Santander, Spain.
2
Institut de Biologia Molecular de Barcelona, CSIC,
Jordi Girona, 18–26, 08034 Barcelona, Spain.
Summary
Bacterial conjugation is a promiscuous DNA trans-
port mechanism. Conjugative plasmids transfer them-
selves between most bacteria, thus being one of the
main causal agents of the spread of antibiotic resis-
tance among pathogenic bacteria. Moreover, DNA can
be transferred conjugatively into eukaryotic host
cells. In this review, we aim to address several basic
questions regarding the DNA transfer mechanism.
Conjugation can be visualized as a DNA rolling-circle
replication (RCR) system linked to a type IV secretion
system (T4SS), the latter being macromolecular
transporters widely involved in pathogenic mecha-
nisms. The scheme ‘replication + secretion’ suggests
how the mechanism would work on the DNA sub-
strate and at the bacterial membrane. But, how do
these two parts come into contact? Furthermore,
how is the DNA transported? T4SS are known to be
involved in protein secretion in different organisms,
but DNA is a very different macromolecule. The so-
called coupling proteins could be the answer to both
questions by performing a dual role in conjugation:
coupling the two main components of the machinery
(RCR and T4SS) and actively mediating DNA trans-
port. We postulate that the T4SS is responsible for
transport of the pilot protein (the relaxase) to the
recipient. The DNA that is covalently linked to it is
initially transported in a passive manner, trailing on
the relaxase. We speculate that the pilus appendage
could work as a needle, thrusting the substrate pro-
teins to cross one or several membrane barriers into
Accepted 8 April, 2002. *For correspondence. E-mail delacruz@
unican.es and llosam@unican.es; Tel. (+34) 942 201 942/57; Fax
(+34) 942 201 945.
© 2002 Blackwell Science Ltd
the recipient cytoplasm. This is the first step in con-
jugation. The second step is the active pumping of
the DNA to the recipient, using the already available
T4SS transport conduit. It is proposed that this
second step is catalysed by the coupling proteins.
Our ‘shoot and pump’ model solves the protein–DNA
transport paradox of T4SS.
Conjugation as the merging of two ancient
bacterial systems
DNA replication and macromolecule transport across
membranes are basic processes of life. If linked, they
could form the basis for a new mechanism, DNA secre-
tion, by simply driving the displaced replicating DNA
strand to the macromolecular transporter in the mem-
brane. It is tempting to imagine that bacterial conjugation
once arose in this way. For this to happen, some DNA-
replicating enzymes on one side and protein transporters
on the other would have to adjust to new substrates. In
addition, a new protein is needed to bring the displaced
DNA in contact with the transporter: a coupling protein.
With this one and only new protein (and a few adjust-
ments), bacteria could have acquired the basics for con-
jugation, a mechanism that provides them with a unique
means for genetic exchange and a powerful source of
genetic variability.
Indeed, bacterial conjugation systems (including the
related Vir system that Agrobacterium tumefaciens uses
to transfer DNA to plants) show striking similarities to both
DNA replication and macromolecular transport systems.
Conjugative DNA-processing enzymes and their sub-
strate DNA sequence (components of the so-called
relaxosome) show extended sequence similarity to
rolling-circle replication (RCR) systems (Waters and
Guiney, 1993). Moreover, the set of conjugative proteins
that assembles the membrane transporter belong to the
type IV secretion system (T4SS) family (Christie, 2001).
In addition to proteins with similarity to RCR or T4SS, all
conjugative systems include one protein that has no
obvious counterpart in either system. This is considered
to be the coupling protein that connects the relaxosome
with the membrane transporter. The evidence for this role
will be discussed below.
2 M. Llosa et al.
Based on the ‘replication + secretion’ scheme, we
can construct a first picture of the conjugative transfer
process. (i) In the cytoplasm, an RCR-like system nicks
the transferred strand (T-strand) at the origin of transfer
(oriT ), unwinds the nicked strand, replicates the remain-
ing strand from the free 3¢-OH end and, once a full-length
T-strand has been displaced, rejoins both ends. Bio-
chemical evidence supports the similarity of the DNA-
processing reactions in both conjugation and RCR (Lanka
and Wilkins, 1995). (ii) The coupling protein guides this
protein–DNA complex to the T4SS at the membrane,
where the T-DNA gets out of the bacteria.
But does DNA exit through the T4SS? T4SS have been
shown to be directly responsible for protein transport in
several systems (see below). It is difficult to imagine how
such a different macromolecule as DNA would use the
same transporter. We propose that the coupling protein,
in addition to its coupling role, serves as an adaptor for
the T4SS to a new substrate, DNA instead of protein,
working as a pushing molecular motor that would confer
processivity to the secretion process.
Finally, the mechanism of entering the recipient cell
remains to be determined. Conjugation implies the cross-
ing of at least one recipient lipidic barrier and usually more
than that. Our proposal is that the pilus assembly opens
the way into the recipient through all barriers.
Coupling proteins
Coupling proteins are present in all conjugative systems.
The best characterized are proteins TrwB of IncW plasmid
R388, TraD of IncF plasmids, TraG of IncP plasmids and
VirD4 of A. tumefaciens Ti plasmids. There are no cou-
pling proteins in mobilizable plasmids, such as RSF1010
or ColE1, with the notable exception of plasmid CloDF13
(Núñez and de la Cruz, 2001). These proteins share the
following features:
(i) They are not involved in DNA processing reactions
(Pansegrau et al., 1990; Howard et al., 1995; Llosa et al.,
1995; Scheiffele et al., 1995; Tinland et al., 1995) or in
pilus production (Bolland et al., 1990; Waters et al., 1992;
Lai et al., 2000). They are needed after pilus assembly
and contact formation (Panicker and Minkley, 1985). Inter-
estingly, traD mutants of plasmid F inhibit the progression
of transfer, but not its start: they prevent DNA transport to
the recipient cell, but not conjugal DNA synthesis in the
donor cell (Kingsman and Willetts, 1978).
(ii) They have several (usually two) transmembrane a-
helices in their N-terminal region that mediate anchoring
to the inner membrane and a main cytoplasmic C-termi-
nal domain (Llosa et al., 1994; Das and Xie, 1998; Lee et
al., 1999; Grahn et al., 2000). Thus, their location is ade-
quate to be the link between a cytoplasmic system and a
membrane complex.
(iii) They include conserved Walker motifs in their
sequence. No NTPase activity has been reported so far,
but the presence of these motifs raises the possibility that
they use ATP hydrolysis as an energy source to work as
motors. A soluble domain of protein TrwB (TrwBDN70)
binds NTPs (Moncalián et al., 1999), and binary com-
plexes with different NTPs or analogues have been crys-
tallized (Gomis-Rüth et al., 2002). Mutants in putative
NTP-binding residues are transfer deficient (Balzer et al.,
1994; our unpublished results).
(iv) Protein TrwBDN70 is able to bind both single- and
double-stranded DNA non-specifically (Moncalián et al.,
1999); TraD and TraG may bind DNA too (Panicker and
Minkley, 1992; E. Lanka, cited by Baron et al., 2002). This
is a key aspect of coupling proteins. They appear to be
mostly associated with systems known to transfer DNA
(see below), suggesting a possible specific role in DNA
transport.
(v) Unlike relaxosome components, which are specific for
their cognate oriT sequences, coupling proteins show
some level of interchangeability (Cabezón et al., 1994).
There is indirect genetic data that coupling proteins may
contact both the relaxosome and the transporter: using
pieces from different conjugative systems, it was shown
that the efficiency of coupling proteins in conjugation
varies depending on both the relaxosome and the T4SS
components used (Cabezón et al., 1997; Hamilton et al.,
2000). In addition, direct interactions of coupling proteins
with relaxosome components have been reported, both in
vitro and in vivo (Disque-Kochem and Dreiseikelmann,
1997; Sastre et al., 1998; Szpirer et al., 2000; F. de la Cruz
and E. Lanka, cited by Baron et al., 2002). No direct proof
exists so far that these proteins interact with the T4SS but,
as T4SS form transmembrane complexes, interactions
may be more difficult to assess.
How would a coupling protein work? Their location and
interactions strongly suggest that these proteins are the
specific factors needed to make the contact between
the relaxosome and the secretion machinery. Also, the
binding of NTP and DNA suggests that the proteins use
the triphosphate energy to pump DNA into the T4SS.
Their role could be to thread the T-DNA towards the trans-
membrane pore and/or motor the DNA through it. As a
main difference between a polynucleotide and a polypep-
tide is the length of the former, it seems that a motor would
be needed to confer processivity to the transporter.
Sequence similarities further support this role. A com-
parison of coupling proteins against protein databases
shows similarities to a family of bacterial proteins involved
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1–8

in DNA transport (Errington et al., 2001). This family
includes proteins FtsK and SpoIIIE, involved in chromo-
some segregation at cell division and sporulation respec-
tively. Both proteins locate to the septum, anchored to the
membrane by their N-terminal domain, and have a main
catalytic cytoplasmic domain, like conjugative coupling
proteins. FtsK is an Escherichia coli division protein
responsible for the specific partition of chromosome
dimers at the septum at a late stage in cell division. It has
been shown recently that FtsK is a DNA motor protein: it
forms multimers that ring around DNA using ATP hy-
drolysis to translocate along it (Aussel et al., 2002). These
authors also show that FtsK activates dimer resolution by
an interaction with XerCD recombinases. Protein SpoIIIE
of Bacillus subtilis is required for chromosome segrega-
tion into the prespore compartment. SpoIIIE has recently
been characterized as a DNA exporter (Sharp and
Pogliano, 2002). Its cytoplasmic domain is a DNA-
dependent ATPase capable of tracking along DNA in the
presence of ATP (Bath et al., 2000). Thus, during sporu-
lation, SpoIIIE appears to act as a DNA pump that actively
moves one of the replicated pair of chromosomes into the
prespore. Finally, a group of proteins from Streptomyces
called Tra proteins also belongs to the FtsK/SpoIIIE
family; these proteins are responsible for intermycelial
transfer of plasmids. All these similarities suggest that
coupling proteins are DNA transporters that could work in
a similar way to FtsK and SpoIIIE, by tracking along the
DNA and thus pushing it through the T4SS. It has to be
noted that FtsK and SpoIIIE act on double-stranded DNA,
whereas coupling proteins would presumably track on
single-stranded DNA.
The only known three-dimensional structure in the
FtsK/SpoIIIE/coupling protein superfamily is that of the
cytoplasmic domain of TrwB (Gomis-Rüth et al., 2001;
2002). This structure has provided valuable information
on possible mechanisms of action for the coupling protein
family. The protein has three structural domains: an N-
terminal trans-membrane domain (TMD) that crosses the
membrane twice and was deleted for structural work; a
nucleotide-binding domain (NBD) that includes the Walker
motifs and similarities with FtsK/SpoIIIE; and an all-alpha
domain (AAD) facing the cytoplasmic side. The AAD
shows significant structural similarity with XerD recombi-
nase. The NBD resembles the RecA core structural fold,
present in proteins such as the F1 subunit of the ATPase
complex and in DNA ring helicases.
In view of the structural analogy, it is reasonable to
deduce that TrwB could work as either a DNA helicase or
a rotary DNA-tracking protein. A DNA helicase would drive
the displaced DNA strand to the transporter as a result of
its unwinding activity. However, TrwB has not yet been
shown to possess DNA-dependent ATPase activity, a sig-
nature of DNA helicases. Alternatively, TrwB could work
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1–8
A two-step model for conjugation 3
in an analogous way to F1 ATPase, pushing DNA directly
into the transmembrane pore through its rotational
tracking along DNA. Sequence similarity to SpoIIIE/FtsK
reinforces this hypothesis.
DNA transport versus protein transport
T4SS are versatile macromolecular transporters. They
are part of varied bacterial processes such as conjuga-
tion, T-DNA transfer to plants and toxin secretion. Lately,
they have become the centre of much attention because
of the finding that many human pathogens, such as
Helicobacter pylori, Brucella or Bartonella, code for T4SS
that are directly involved in virulence (recently reviewed
by Christie, 2001). These processes imply protein and/or
DNA secretion. However, DNA and proteins are very dif-
ferent macromolecules. How could they both be trans-
ported in a similar way? Is there evidence that both
proteins and DNA are substrates for T4SS?
T4SS are protein secretors in many systems. The Ptl
T4SS of Bordetella pertussis is responsible for secretion
of the pertussis holotoxin (Farizo et al., 2000). Most of the
newly discovered T4SS in pathogens are thought to be
protein transporters, but the actual substrate is unknown,
except in H. pylori, which uses a T4SS to translocate
protein CagA into eukaryotic cells (Odenbreit et al., 2000).
In conjugative systems, DNA is known to be transported
into the recipient bacteria. Proteins may be translocated
too: in certain conjugation systems, it has been shown
that a DNA primase gets into the recipient cell (Rees and
Wilkins, 1990; Wilkins and Thomas, 2000). We hypothe-
size that the protein that initially nicks the T-strand, the
relaxase, is translocated with the DNA as a pilot protein
into the recipient cell where it would perform the final
strand-transfer step. Then, the relaxase would be the true
substrate of conjugative T4SS. In the case of the Ti
plasmid of A. tumefaciens, the function of VirD2 as a pilot
protein is clear, and other Vir proteins are transported into
plant cells (Vergunst et al., 2000) in addition to the T-DNA.
Although no conjugative relaxase has yet been shown to
be transported, this could be due to experimental difficulty,
as presumably a single relaxase molecule would get into
the recipient, whereas in the donor cell there can be hun-
dreds of them (Abdel-Monem et al., 1977; G. Grandoso
and F. de la Cruz, unpublished results).
Our hypothesis is that, after relaxase transport, the cou-
pling protein would be the specific factor that adapts a
T4SS to DNA secretion. A strong argument in support of
this idea is the already mentioned similarity between cou-
pling proteins and FtsK/SpoIIIE, which are known DNA
transporters. Other arguments are that, on the one hand,
no coupling protein is found associated with the Ptl
system, which strongly suggests that a T4SS may be able
to secrete proteins without a coupling protein. On the
4 M. Llosa et al.

other hand, no DNA secretion has been shown so far in
the absence of a coupling protein.
Against this hypothesis, mutational analysis showed
that coupling factors are required for protein secretion in
both H. pylori (for CagA translocation; Fischer et al., 2001)
and the Ti plasmid Vir system (for VirF and VirE2 secre-
tion; Vergunst et al., 2000). However, this does not rule
out a main role for coupling proteins as DNA transporters;
if the couplers were originally recruited to secrete DNA
by processes that need to secrete proteins too, their
posterior involvement in protein secretion could help in
the synchronization of the overall process. Besides, it is
noteworthy that the coupling protein of the H. pylori T4SS
binds DNA (E. Lanka, cited by Baron et al., 2002). In addi-
tion, two putative DNA relaxases have been found in the
H. pylori genome (Backert et al., 1998). Thus, DNA
translocation (present or past) cannot be ruled out in
H. pylori.
A two-step model for DNA transport
We would like to present a model for conjugal DNA trans-
port based on the above proposed roles for coupling pro-
teins and pilot proteins, using the plasmid R388 transfer
system as a paradigm. In R388, the coupling protein is
TrwB, of known three-dimensional structure, and the pilot
protein is TrwC, which has relaxase and DNA helicase
activities (Grandoso et al., 1994; 2000; Llosa et al., 1995;
1996). We propose that the T-strand is transported in two
mechanistically distinct steps (Fig. 1). In the first step, the
DNA would be transported through the T4SS in a passive
form, as a tail that is covalently linked to the pilot protein
TrwC, the active substrate for the T4SS. After this step,
the situation would be similar to that of the septum formed
in division/sporulation. In a second step, TrwB would
pump the T-DNA processively into the T4SS, in much the
same way as FtsK or SpoIIIE pump the DNA to the divid-
ing cell or the spore respectively. This two-step mecha-
nism explains the need for the coupling protein late in the
conjugation process, as well as most of the features of
coupling proteins outlined above.
The overall process could be viewed as follows: a TrwC
dimer binds oriT; one monomer nicks the T-strand and
remains covalently bound to the 5¢ end. The relaxosome is
in close contact with the transporter through TrwB. Upon
detection of a mating signal (that might come through the
membrane, inducing a change in TrwB or in any of the
relaxosome components), TrwC starts unwinding and the
dimer dissociates; the T-DNA-bound monomer exits
through the T4SS, carrying the T-strand along; the TrwC
monomer remaining in the donor continues to unwind the
DNA and performs the second strand-transfer reaction
that liberates the T-strand from the donor molecule upon
reaching the nic site for a second time. The displaced DNA
strand, at first passively stuck within the T4SS that has
actively transported the relaxase, is then pumped out by

Fig. 1. A two-step model for conjugal DNA transport. Horizontal thick black lines represent bacterial membranes, traversed by grey cylinders
that represent the T4SS. TrwC is represented as the two-domain circle + oval (relaxase + helicase) shape; TrwB is represented as a hexamer,
with an orange-like shape, anchored to the inner membrane. DNA is represented by a thin black line; newly replicated DNA, by a dashed arrow.
The vertical arrowhead represents the nic site. Curved arrows indicate postulated motion forces required for DNA movement.
A. TrwB is coupling the T4SS and the relaxosome; a TrwC monomer covalently linked to the nicked T-strand is the substrate for T4SS
secretion.
B. TrwB is pumping out the T-strand as it is displaced from the donor plasmid. Upon reaching the nic site for the second time, the TrwC
monomer in the donor would perform a second strand-transfer reaction, thus liberating the T-strand. The translocated TrwC monomer would
rejoin the two T-strand ends by a reverse cleavage reaction.

© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1–8


TrwB, which tracks along it; this is the motive force that
sends the DNA strand through the transporter and into the
recipient. In view of the recent evidence on the homologue
FtsK (Aussel et al., 2002), the most plausible scenario is
that the DNA goes through the central channel in the TrwB
hexamer. Once the T-strand is completely transferred, the
TrwC monomer in the recipient, bound to the 5¢ end, would
transfer it to the free 3¢ end.
Other alternatives cannot definitely be ruled out at the
present time. It remains possible that the T4SS is strictly
a protein transporter and DNA moves through a separate
pathway. After all, no direct interaction has ever been
reported between coupling proteins and T4SS, and there
is no definite proof for DNA going through it. In fact,
protein secretion through the T4SS has been shown to
occur independently of DNA transport in both A. tumefa-
ciens (Vergunst et al., 2000) and conjugation (Wilkins and
Thomas, 2000).
Another possibility would be the existence of a periplas-
mic step in secretion, which has been proposed for the
Ptl system based on the recent finding (Farizo et al.,
2002) that the holotoxin must be assembled in the
periplasm for efficient secretion. Ti plasmid Vir proteins
secreted through their cognate T4SS have also been
detected in small amounts in the periplasm (Chen et al.,
2000).
Finally, our model is based on coupling proteins working
as motors. However, it is also possible that these proteins
do not hydrolyse NTPs as an energy source to pump
DNA. In R388, TrwC has an ATPase activity that is used
to unwind DNA. This could be an alternative driving force
to push the T-strand into the transporter. In this case, the
role of the coupling protein would be threading the DNA
into the T4SS rather than actively pumping it.
Crossing all barriers
Most research efforts in bacterial conjugation have been
addressed to finding out how DNA is first processed in the
cytoplasm and then exits the donor bacterium. Almost
nothing is known about it entering the recipient cell. In
conjugation-like mechanisms, there are two to four lipidic
barriers to cross, apart from other barriers such as
lipopolysaccharides or peptidoglycan cell walls. How are
they all trespassed?
Conjugation was ‘designed’ to work among bacteria.
Curiously, however, the same transfer system that oper-
ates between two bacteria works to get T-DNA into other
cell types as different as yeast, plant or mammalian cells
(Buchanan-Wollaston et al., 1987; Heinemann and
Sprague, 1989; Waters, 2001). Thus, the mechanism of
entry cannot be host specific. It appears that conjugation
can transport DNA almost regardless of the number and
sorts of barriers in its way. Maybe we should envisage this
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1–8
A two-step model for conjugation 5
process as more aggressive than we traditionally think,
that is working as a needle that can perforate all kinds of
biological membranes.
Conjugative systems (including the Ti plasmid Vir
system) form a filamentous appendage called pilus.
Traditionally, the pilus was believed to play a role in at-
tachment to the recipient. There was a long debate
regarding the possibility that the pilus itself worked as a
channel inside which the DNA would travel from one cell
to the other. This possibility has not been completely ruled
out, although it is not favoured by most experts. The
recent finding that there are Ti plasmid Vir mutants that
do not produce pili, yet they transfer DNA (Sagulenko et
al., 2001), argues against a role for the pilus as a DNA
channel.
Not all T4SS have been shown to be associated with a
pilus. The only known T4SS that does not imply cell-to-
cell transport, the Ptl system for pertussis toxin secretion
into the milieu, does not have a pilus. No pili have been
found so far associated with T4SS present in bacteria that
infect animal cells. On the other hand, pili have been
detected associated with type III secretion systems
(T3SS), specifically in those that plant pathogens use to
deliver virulence proteins into plant cells. These pili are
not involved in attachment to plant cells but are the actual
channels for protein transport (Jin and He, 2001).
However, no pili have been detected associated with
other T3SS such as that of Yersinia, intended to inject
virulence proteins into animal cells. These systems form
other much shorter structures that have been called
‘needles’, which are the actual conduits for protein trans-
port into the recipient animal cell (Hoiczyk and Blobel,
2001).
Regardless of the type of secretion system, the
secreted substrate or other possible functions in attach-
ment or transport, it seems that pili are associated with
systems intended to reach the cytoplasm of either plants
or Gram-negative bacterial cells, that is recipients that
present additional barriers in their envelopes when com-
pared with the single lipidic bilayer present in animal cells.
As suggested previously (Llosa and Zambryski, 1998), pili
could be required for opening the way through whatever
additional barriers/envelopes are present in the recipient,
thus allowing the entrance of DNA and/or proteins through
a bacterial secretion system. T3SS or T4SS would be the
syringes for macromolecular injection, and pili would work
as the needles. Another possibility, either as an alterna-
tive to the idea that the pilus opens the way by brute force
or in addition to it, is that the tip of the pilus would have
some enzymatic activity that could digest peptidoglycan
or plant cell walls.
Apart from opening the way, pili could inject proteins
directly into the recipient cell by their sheer assembly
force. Proteins could be carried at the pilus surface as it
6 M. Llosa et al.
grows, as in the ‘external conveyor belt’ model proposed
for the Hrp pilus (Jin et al., 2001). In the Ptl system,
although there are not assembled pili, there is a pilin
homologue; it has been suggested (D. Burns, personal
communication) that this pilin could push out the holo-
toxin, as in the beginning of a growing pilus. This could
also be the mechanism for protein translocation into other
cells, but with the help of the assembled rigid pilus to pen-
etrate the recipient. A similar mechanism has been pro-
posed for T2SS (Sandkvist, 2001). The appendage
needed to penetrate a recipient animal cell would be
much shorter than the one needed to penetrate a bac-
terium or a plant cell, thus appendages of different lengths
could be assembled in each case, like the needles and
pili found associated with T3SS in animal- and plant-
associated bacteria respectively. In conjugation, the pilot
protein could be injected directly into the recipient’s cyto-
plasm with the covalently linked T-strand. The coupling
protein would then push the rest of the DNA along the
same way.
In summary, we visualize conjugation as a two-act
drama. In the first act, a T4SS shoots the pilot protein and
the DNA that lags behind to the recipient, perforating one
or several membranes on its way. In the second act, the
bulk of the DNA is pumped to the recipient cell by the cou-
pling protein molecular motor. This ‘shoot and pump’
model solves the protein–DNA transport paradox of T4SS.
Besides, we propose specific functions for T4SS and cou-
pling proteins in conjugation-like mechanisms.
Acknowledgements
This work was supported by the Ministerio de Ciencia y Tec-
nología (Spain) (grants PB98-1106 to F.C., PB98-1631 and
2FD97-0518 to M.C. and BIO2000-1659 to F.X.G.-R.), and
the Generalitat de Catalunya (grant 1999SGR188 to M.C.).
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