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,*
1
Institut de Biologia Molecular de Barcelona, C.S.I.C., c/ Jordi Girona, 18-26, E-08034 Barcelona (Spain) and
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)
2
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 extracellular 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
1552
Current Pharmaceutical Design, 2004, Vol. 10, No. 13
genes between different species and even for trans-kingdom
gene transfer [25-30]. Conjugative DNA transfer depends on
genes contained in the plasmid transfer (tra) region, further
divisible into dtr (from DNA transfer and replication) and
mpf (from mating-pair formation; [26]). Dtr proteins process
conjugative DNA through formation of the relaxosome, a
nucleoprotein complex. This implies sequence-specific
recognition of a cis-acting DNA sequence called origin of
transfer (oriT) and a DNA site- and strand-specific cleavage
by specialised nicking enzymes, or relaxases, that hydrolyse
a single phosphodiester bond and attach covalently to the
newly created 5’ terminus. This process is followed by a
strand displacement reaction, which generates a singlestranded (ss) DNA transfer intermediate, the T-strand [21]. A
simultaneous rolling-circle DNA replication reaction is
observed for replacement of the displaced strand in the donor
cell (Fig. (1a); [30]). The relaxosome is attached to the
transport site facing the cytoplasmic side of the cell envelope
[31] and the DNA-transfer channel complex, a T4SS formed
by 11-12 plasmid Mpf proteins [26], transports the T-strand
to the recipient cell with 5’ to 3’ polarity. Conjugative
systems, an example is Escherichia coli F sex factor conjugation [32], may also deliver non-self-transmissible plasmids
to recipient cells [25, 33] and translocate DNA even across
kingdom barriers, e.g. to Saccharomyces cerevisiae [29].
Furthermore, these systems may shuttle proteins to recipient
cells independent of DNA, as shown for RecA protein during
the transfer of plasmid RP4 to E. coli recipients [34].
In the soil phytopathogen Agrobacterium tumefaciens,
the type-IV VirB transporter translocates a segment of the
tumour-inducing 200-kb Ti plasmid DNA, the T-region, in
the form of an ss nucleoprotein T-complex with the
covalently attached VirD2 protein from the bacterium to
dicotyledonous plant cells. This results in phytohormone
overproduction and crown-gall disease in the latter [1, 3538]. This translocon has been exploited in plant biotechnology as a shuttle to introduce new genes conferring
resistance 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 as
a transporter [20]. The process requires additional protein
translocation into the plant cell, so as precise cleavage and
cyclisation of pilin-like protein VirB2 [35, 40]. The VirB
system also mediates a conjugation-like transfer of suitable
plasmids to recipient plant and bacterial cells [25, 37] and of
T-DNA to the three major groups of fungi, the molds, the
yeasts, and the mushrooms, as shown for S. cerevisiae,
Aspergillus niger, Fusarium venenatum, Trichoderma reesei,
Colletotrichum gloeosporioides, Neurospora crassa and
Agaricus bisporus [15, 21, 41]. A recent report even demonstrated DNA transfer to human cells [42]. A. tumefaciens Ti
plasmid codes for a second transfer system responsible for Ti
conjugative transfer between bacteria. This process operates
through a functionally and physically independent tightlyregulated tra-system, containing tra and trb genes, which are
equivalent to dtr and mpf genes, respectively [36]. A third
system is featured by avhB encoded proteins, promoting
conjugal transfer [43]. Furthermore, a chromosomal tra-like
region has been recently identified in an Ti-free A.
tumefaciens strain, raising the question of the role of such a
chromosomal region during evolution [44].
Gomis-Rüth et al.
Some microbial facultative intracellular pathogens,
which withstand bactericidal activities of polymorphonuclear
cells, survive within professional phagocytes and multiply
inside the phagosome [45]. They live in intracellular
vacuoles until the cell lyses and releases bacteria that start
new rounds of infection [46]. These pathogens possess a
T4SS believed to export macromolecules to the vacuolar
membrane or to the cytoplasm of the host cell, which adapt
the vacuolar environment to the bacterial needs, allowing
intracellular survival and replication [15, 16, 47]. Brucellosis
or Malta fever provokes chronic infections and bacteremia in
mammals, including humans, and is caused by Brucella suis,
abortus and melitensis [17, 48, 49]. Their genomes also
encode 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 by
phagosome acidification in cells after phagocytosis. A recent
report on the genome sequence of typhus-causing Rickettsia
prowazekii [53] has revealed seven homologues of VirB
proteins of A. tumefaciens, but not of the T-DNA-bound
VirD2 and VirE2 proteins. Legionella pneumophila, the
cause of Legionnaire’s disease, a form of lobar pneumonia
[54, 55], contains four icm/dot genes with significant
sequence similarity to A. tumefaciens Vir proteins and those
of various conjugal transfer systems [56]. This system is
essential for apoptosis induction in human macrophages [57]
and for secretion of protein DotA [58]. As reported for the
VirB transporter, the icm/dot system transfers E. coli
RSF1010 plasmid between bacteria, probably as a
nucleoprotein complex [33, 59, 60]. Like A. tumefaciens, L.
pneumophila encodes a second type-IV translocon, not
involved in pathogenesis, containing 11 lvh genes arranged
similarly to the icm/dot genes. This apparatus is dispensable
for cellular growth and it can replace some components of
the icm/dot system to enable RSF1010 conjugation [61].
Most recently, Ehrlichia phagocytophila and chaffeensis, the
ethiological agents of granulocytic and monocytic
ehrlichioses 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 toxic
proteins. These systems may employ the Sec system for
protein translocation across the inner membrane and prior to
secretion [9, 63, 64]. In Helicobacter pylori , the microorganism responsible for gastritis, peptic ulcer and gastric
cancer, a pathogenicity island (cagPAI), acquired by
horizontal transfer, is the major determinant of virulence.
This island codes for 31 Cag proteins, at least six of which
may contribute to a type-IV system, which translocates in an
example for direct trans-kingdom protein delivery the 150kDa CagA antigen into gastric epithelial cells, where it is
tyrosine phosphorylated [65, 66]. There, the protein may
interfere with the normal host cell signalling inducing
pedestal formation, synthesis of proinflammatory cytokines
and chemokines, like interleukin 8, cell spreading and
motiliy, expression of the protooncogenes c fos and c jun,
among other events [16, 67, 68], in a phenotype referred to
as “hummingbird”. It occasionally enters epithelial cells and
manipulates the host immune system for immune evasion
Coupling Factors in Macromolecular Type-IV Secretion Machineries
Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1553
[69]. Like A. tumefaciens, this pathogen possesses a conjugation-like mechanism of DNA transfer [69-72]. In the closely
related Helicobacter hepaticus, three basic components of a
T4SS have been found on a genomic island, suggesting the
presence of an orthologous system [73]. Yersinia enterocolitica
29930, producing the phage-tail resembling bacteriocin
enterolicin harbours a T4SS encoded on a plasmid. It is a
conjugative transfer system closely related to the Mpf system
of plasmid R6K. However, an orthologue to TaxB of the
latter system is missing [74]. Finally, Bordetella pertussis,
the causative agent of whooping cough in humans, has coopted a set of conjugal transfer system genes, ptl (from
pertussis toxin liberation) [75]. Ptl proteins are highly similar
to VirB proteins and direct the export of the A-B-type
endotoxic Pertussis toxin, composed of six subunits, across
the bacterial envelope and into the medium [21].
Additional candidate type-IV translocons, or at least
some constituents, can be found in pathogenic and nonpathogenic organisms and in reported bacterial genome
sequences (Table 1).
TRANSFER FACTORS IN CONJUGATIVE TYPE-IV
Table 1.
SECRETION
The described type-IV transport systems require, in addition to the apparatus directly responsible for macromolecular
trafficking, an aiding transfer factor or coupling protein [76,
77], when performing conjugation-like nucleoprotein
transfer. Such a protein is encountered in all selftransmissible plasmids of Gram-negative bacteria and in
many of Gram-positive bacteria [26]. There is strong
evidence that these proteins are also required for type-IV
mediated protein export, as reported for A. tumefaciens
VirE2 and VirF protein export (in addition to the T-strand /
VirD2 complex) and H. pylori [14, 16, 21, 35, 78]. On the
other hand, type-IV systems responsible for only protein
export, like the Bordetella Ptl system, may lack T4CPs.
This class of coupling proteins are integral innermembrane proteins, with an N-terminal transmembrane part
and a C-terminal cytoplasmic moiety, that may assemble as
components of the cognate type-IV transport machinery or
may be required to localise near the transport apparatus [7987]. They bear Walker A (motif I) and B (motif II or DExx
box) NTP-binding sequences [88] and may use the energy of
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]
1554
Current Pharmaceutical Design, 2004, Vol. 10, No. 13
NTP hydrolysis to connect transmissible (nucleo)protein
complexes with the cognate translocation system [24, 67, 76,
82, 84, 89-92]. Mutational studies have revealed that inactive
T4CP mutants affecting NTP binding and/or hydrolysis lead
to conjugation-deficient phenotypes [24, 79, 82, 83]. Conjugation inhibition may also occur when T4CPs associate with
other proteins, as shown for RP4 TraG when it interacts with
FopA of pKM101 and PifC of plasmid F [89]. Studies on
chimeric systems comprising the transport apparatus of one
species and the T4CP of another reveal that specificity
resides on the latter [93, 94]. In some cases, as found for
TraD from F, their C-terminal parts affect efficiency and
specificity [95].
TrwB from E. coli plasmid R388, TraD and TraG
proteins from various Gram-negative plasmids, VirD4 from
A. tumefaciens Ti and related proteins are T4CPs associated
with nucleoprotein mobilisation (Table 2). They can be
unambiguously aligned using the hidden Markov model
method SAM T99 [96] and they are divisible into subfamilies. The first, which includes TrwB and TraD, contains six
members of conjugative plasmids. A second subfamily
groups a broad set of conjugative plasmids (like TraG of
RP4), and the VirD4 protein of Ti plasmids involved in TDNA transfer to plant cells (Table 2). Proteins within a
subfamily are >30% identical in amino acid sequence and
display the same domain organisation (Fig. (1b)). In particular, TrwB displays 21-23 % sequence identity to VirD4 of
A. tumefaciens Ti [19] and TraG of RP4 and R751, and 2830% to TraD of F and R100 [84, 85]. The orthologue from
plasmid R3888 binds both ss and double stranded (ds) DNA
with approximately the same affinity as supercoiled dsDNA.
This DNA binding ability is unspecific and independent of
NTP binding. TraD from plasmid F, TraG from RP4 and
HP0524 from H. pylori have also been reported to bind ss
and dsDNA non-specifically [81, 97]. None of these
orthologues displayed detectable NTP-hydrolysing activity
in vitro under the conditions assayed. TrwB is inserted on
the cytoplasmic side of the inner membrane, as described
also for TraD and VirD4 [79-81, 87]. The latter has a unique
cellular location to the donor cell pole and both its
periplasmic and the cytosolic parts are required for polar
localisation, indicating where the associated type-IV
secretion system may also be situated [79]. Some of these
proteins can be functionally exchanged for each other. Thus,
TraG can substitute TrwB in the mobilisation of plasmids
ColE1 and RSF1010 by R388, but not in self-transfer of
R388 [93]. Evidence has also been found for the interaction
partners of T4CPs. TraG from RP4 interacts with the
relaxosome relaxase [92, 97]. In plasmid F, TraD contacts an
accessory nicking protein, TraM [98], and in plasmid R27,
TraG interacts with the T4SS protein TrhB [99].
T4CPS AND SPOIIIE / FTSK PROTEINS
T4CPs and members of the SpoIIIE/FtsK protein family
share functional and structural features (Fig. (1b); [82, 100102]). SpoIIIE is an essential sporulation membrane-bound
protein in Bacillus subtilis, whose cytoplasmic part interacts
with the chromosomal DNA to achieve DNA translocation
from the mother cell through an annulus to the prespore. It is
Table 2.
Gomis-Rüth et al.
also responsible for the polarity of DNA transfer [103].
Several orthologues of SpoIIIE have been reported in
Bacillus halodurans, Sporosarcina ureae and Streptomyces
coelicolor. SpoIIIE displays significant sequence similarity
to Tra proteins of several Gram-positive conjugative plasmids of Streptomyces [101]. The annulus may be lined up by
a number of SpoIIIE proteins to render a continuous ring,
through which the prespore chromosome may translocate
[104]. The C-terminal cytoplasmic transfer domain of
SpoIIIE shows ATPase activity and the protein can track
along DNA in the presence of ATP [100, 102]. The protein
behaves as a DNA pump that actively moves one of the
replicated pair of chromosomes into the prespore [100]. On
the other hand, E. coli FtsK is involved in the resolution of
chromosomal dimers during bacterial cell division [105]. It
precisely synchronises the separation of the two daughter
chromosomes by XerCD recombinase through site-specific
recombination [106]. FtsK may also pump newly replicated
chromosomes away from the septal space during septation
[105]. Orthologues of FtsK, which is essential, are probably
present in all bacteria (to date reported for Campylobacter
jejuni, Coxiella burnetii, Haemophilus influenzae, Proteus
mirabilis, H. pylori, Azospirillum brasilense, Chlamydia
trachomatis, muridarum and pneumoniae, S. coelicolor,
Pseudomonas aeruginosa and syringae, Neisseria meningitides, R. prowazekii, Deinococcus radiodurans, Ureaplasma
parvum and Vibrio cholerae), but also in eukarya (Caenorrhabditis elegans). The SpoIIIE/FtsK analogues found to
date are YPTP from B. subtilis, Tra/KilA from Streptomyces
lividans plasmid IJ01 and Trasa/Integrase fusion protein,
among others. T4CPs have the same molecular organisation
as the C-terminal domain of SpoIIIE/FtsK-like proteins (Fig.
(1b)) and share extensive sequence similarities, especially
around the NTP-binding Walker motifs. This suggests a
functional link, as an NTP-dependent pump that transfers
nucleoprotein complexes or proteins via type-IV translocons.
THE FOLD AND QUATERNARY STRUCTURE OF A
T4CP
The 33-kb plasmid R388 [107] contains genes that code
for sulphonamide and trimethoprim resistance [108, 109]. Its
mpf region encodes a functional type IV secretion system,
featuring 11 trw genes (trwD to trwN). R388 displays a short
dtr region made up by oriT, trwA, trwB, and trwC [85, 110].
TrwC acts as a relaxase and helicase. It is responsible for
both nick-cleavage at oriT and T-strand unwinding, and its
three-dimensional structure in complex with oriT DNA has
been solved [111-113]. TrwA is a small-sized tetrameric
protein, which binds two sites at oriT and enhances TrwC
relaxase activity, while repressing transcription of the
trwABC operon [114]. These two proteins constitute,
together with oriT DNA and the host-encoded integration
host factor (IHF), the relaxosome of plasmid R388 [85]. IHF
has a modulator role in conjugation [115], as it inhibits
TrwC nic cleavage, probably by affecting the topology of the
TrwC DNA target site [116].
The third R388 dtr protein is TrwB, the T4CP of this
secretory system [82, 85]. The structure of the cytoplasmic
part of TrwB can be divided into an all-α domain and a
Some Members of the Type-IV Coupling Protein (T4CP) Family
Coupling Factors in Macromolecular Type-IV Secretion Machineries
Name
TrwB
TraD
TraJ
VirD4
LvhD4
TraG
TraG
TraK/VirD4-like
TraN/VirD4-like
TrsK
TaxB
Cag524/HP0524/Cag-β
IcmO/DotL
MobC
MobB
Hypot. Protein SCE2.07
TrbB
Orf6
VirD4-like protein
VagA
VirD4-like sex factor protein
(Table 2) contd….
Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1555
Organism
(p=plasmid)
Data Bank access code
(GB, GenBank; SP, SwissProt/
TrEMBL; PIR, Prot.Inf.Res.)
Reference
Escherichia coli R388
Xanthomonas axonopodis pXAC64
E. coli R100
E. coli F
Sphyngomonas aromaticivorans pNL1
pKM101
pCU1
SP Q04230
SP Q8PRK2
SP P22708
SP O87742, P09130
SP 085919
SP O06958
SP Q9X4G1
[82,85]
[171]
[172]
[173]
[174]
[175]
[176]
Agrobacterium tumefaciens/rhizogenes Ti
A. tumefaciens/rhizogenes Ri
Wolbachia sp. wKueYO and wTai
Rickettsia prowazekii
Rickettsia conorii
Legionella pneumophila
E.coli RP4
E. coli R751
E. coli R64
SP P09817, P18594, Q8VT85, Q8VLK3
SP P13464, Q9F585
SP Q9KW36, Q9KW43
SP Q9ZDNA
SP Q92IM9
SP Q9RLR2
SP Q00185
SP Q00184
GB AB027308
[19]
[177-179]
[168]
[53]
[180]
[61]
[31]
[181]
[182]
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
SP O25252
SP Q9PHI9
SP Q9F254
SP Q9F531
SP P55421
SP Q98NT6, Q981R7, GB CAD31437
SP Q44346
GB NP_066690
SP Q8XW89
SP Q937B8
GB AAG38037
SP Q8VRC7
SP Q97HP1
SP Q9IUR9
SP Q07713
SP O87219
GB AAF72343
SP O32582
SP O25260, Q9ZLV3, Q9JMY0, GB
AAF80195
PIR T18329, SP O54524
SP Q9ZF54
SP P08098
PIR T36167
GB NP_052502
SP Q9WW76
GB AAM37472
GB AAM41759, AAM42732
SP Q91UW5
SP Q8RPL3
SP Q8RPL9
GB CAD31465
SP Q93TC4
–
[183]
[184]
[185]
direct submission
[186]
[187]
[188,189]
direct submission
[190]
direct submission
[191]
direct submission
[192]
[193]
[194]
[195]
[196]
[197]
[16,183]
L. pneumophila
Bacteroides fragilis
Enterobacter cloacae CloDF13
Streptomyces coelicolor
ColIB-P9
Pediococcus pentosaceus pMD136
X. axonopodis
X.campestris
E. coli pIPO2T
Anaplasma phagocytophylum
Ehrlichia chaffeensis
R. loti
X. campestris
L. lactis 712
[60]
[198]
[199]
direct submission
direct submission
[200]
[171]
[171]
[201]
[47]
[47]
[153]
direct submission
[202]
1556
Current Pharmaceutical Design, 2004, Vol. 10, No. 13
Name
TrsK-like protein
TrsK-like protein
TraG-like protein
Organism
(p=plasmid)
Putative transposase
?
?
Rhodococcus equi p33701
Bacillus anthracis pXO2
Sulfolobus sp. pINE1
Sulfolobus sp. pNOB8
Klebsiella pneumophila pR55
Salmonella typhimurium
L. pneumophila
?
Mycobacterium tuberculosis
Gomis-Rüth et al.
Data Bank access code
(GB, GenBank; SP, SwissProt/
TrEMBL; PIR, Prot.Inf.Res.)
GB NP_066787
GB NP_053171
GB AF233440
SP O93672
SP Q8VW38
gnl|WUGSC 99287|stmlt2-.Contig1522
gnl|CUCGC 446|lpneumo MF473.111897(different from LvhD4)
gnl|WIGSC 573|kpneumo B
KPN.Contig1729
Reference
[203]
[204]
[205]
[206]
[207]
Searches performed against SwissProt/TrEMBL at www.expasy.ch and against GenBank and PIR at www.ncbi.nlm.nih.gov:80/entrez. Similar domains were looked for with
PRODOM2000.1, 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 a
recipient cell (light green contour) lacking this genetic element. Intercellular contact is mediated by the pilus and type-IV secretory proteins of
the plasmid mpf region. A mating signal, putatively generated by this contact between the donor pilus and the receptor, apparently leads to a
specific interaction between the relaxosome and the T4CP at the inner face of the conjugative pore [32, 92, 97]. The supercoiled dsDNA
plasmid interacts via its oriT region with proteins of the plasmid dtr gene locus creating the relaxosome. ‚ The pilus putatively retracts to
permit intimate contact between bacterial cell walls. The connecting pore or tunnel is formed as a result of mating-pair stabilisation. ƒ The
DNA-strand transferase / helicase or relaxase enzyme of the relaxosome introduces a strand-specific (T-strand) nick at oriT. The relaxosome
interacts with the inner-membrane-anchored T4CP. „… Transfer of the T-strand through the tunnel is putatively initiated, and both generated
ssDNAs replicate following the rolling circle mechanism (dotted lines). † Once the transfer is finished, the free ends of the plasmids are
joined and the pore closes, the acquired surface exclusion disrupts aggregation, and mating bacteria separate. Finally, the identical plasmids
supercoil. Both resulting cells are now capable of a subsequent conjugation event. (b) Schematic alignment of some representatives of the
T4CP family with their length in amino acids. The first subfamily is exemplified by TrwB (plasmid class IncW) and TraD (IncF). A second
subfamily 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) are
represented, separating short periplasmic regions (left) from large cytosolic ones (right). These are mainly made up by the NBDs (dark
brown) with the A and B walker motifs forming the nucleotide-binding site indicated. The all-α domains (light brown) present in the TrwB
and VirD4 subfamilies do not share significant sequence similarity, although they are both inserted into their respective NBDs. (c) Model
depicting a possible arrangement of the 11 mpf encoded Trw proteins (TrwD-N) making up the type-IV translocon engaged in T-strand
delivery from the donor cell to the recipient cell during conjugation of R388. The arrangement is based on the proposed locations for the
homologous VirB proteins [2, 11, 15, 16, 19, 21, 40, 71, 85, 86, 209].
Coupling Factors in Macromolecular Type-IV Secretion Machineries
nucleotide-binding domain (NBD) attached to the inner
membrane, while the first seventy N-terminal residues
feature two transmembrane helices flanking a small
periplasmic region in between ([2, 117]; Figs. (1b) and (2a)).
The NBD features an α/β P-loop-containing NTP hydrolase
core. On the membrane-proximal edge of this central βsheet, a small three-stranded antiparallel sheet is placed,
almost perpendicularly. Its constituting strands are flexible,
owing to the absence of interactions with the transmembrane
domain preceding strand 1 (excised in the experimental
structure; [117, 118]), which has been tentatively modelled
(Fig. (2a)). At the tip of the NBD, at the membrane-distal
part, 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 in
height and 110 Å in diameter (Fig. (2c)). This is consistent
with biochemical data for the native protein migrating as a
hexamer in gel filtration column chromatography (our
unpublished results). A central channel runs from the cytosolic surface (made up by the upper all-α domains) to the
membrane-proximal pole (formed by the NBDs), ending at
the transmembrane pore shaped by the transmembrane
segments (Figs. (2a, b)). This channel is limited to a
diameter of ∼7-8 Å at its cytoplasmic side. This is the closest
point of the channel that, at its membrane end, has an
opening of ∼22 Å.
STRUCTURAL SIMILARITIES
The family of AAA proteins, described for eukaryotes,
prokaryotes and archaebacteria, includes ATPases associated
with a variety of cellular activities that display an NBD with
a fold similar to TrwB NBD. It is an essential family of specialised, chaperone-like enzymes that participate in membrane
fusion and trafficking, organelle biogenesis, proteolysis and
protein folding [119, 120]. It includes the δ’ subunit of the
clamp loader complex of Escherichia coli DNA polymerase
III (Protein Data Bank access code (PDB) 1a5t; [121]), Nethylmaleimide-sensitive fusion protein domain 2, a
cytosolic ATPase required for intracellular vesicle fusion
reactions (PDB 1d2n; [122]), AAA ATPase p97, involved in
homotypic membrane fusion (PDB 1e32; [123]), and ATPdependent protease HsIU-HsIV (PDB 1e94; [124]).
RNA and DNA helicases are molecular motors involved
in DNA metabolism using the energy from NTP hydrolysis
for nucleic-acid unwinding or strand separation and
translocation [125]. Despite a substantial body of biochemical and structural data, it is only in the past couple of
years that this information has unveiled parts of the working
mechanism [126]. The helicases that unwind dsDNA at the
replication fork are ring-shaped hexamers that catalyse the
displacement of the complementary strand [127, 128]. Other
proteins known as RNA helicases use the chemical energy of
NTP hydrolysis to remodel the interactions of RNA and
proteins [129]. The recently reported crystal structures of
two toroidal DNA ring helicases, those of the T7 phage gene
4 protein [130] and RepA [131], suggest a sequential NTPhydrolytic mechanism. E. coli RecA, whose hexameric
architecture was first described by electron microscopy
studies [132], is the structural prototype for these enzymes
[132-134]. As a key ATP-dependent exchange factor, it is
Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1557
essential to genetic recombination and repair. Extensive
structural studies, including several complexes, have been
carried out with monomeric 3’-5’ PcrA DNA helicase, a
protein 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 with
the equivalent part of RecA (PDB 2reb) and other RecA-like
core encompassing enzymes, such as the replicative
helicase/primase of bacteriophage T7 (PDB 1cr0; [127]),
besides to T7 gene 4 ring helicase (PDB 1e0j; [130]) and
PcrA DNA helicase (PDB 2pjr and 3pjr; [136]). Finally, the
recently solved six-clawed grapple-shaped structure of
homohexameric H. pylori Cag525/HP0525 traffic ATPase,
an inner-membrane associated part of the bacterial type-IV
secretion 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-like
proteins display, in addition to a NBD, an all-α domain, as
shown for RecA, AAA ATPases, PcrA DNA helicase, and
the α and β subunits of F1-ATPase, among others, although
arranged in distinct ways with respect to their NBDs.
However, the TrwB all-α domain bears significant structural
similarity only to N-terminal domain 1 of the site-specific
recombinase, XerD, of the λ integrase family (PDB 1a0p;
[139]). This domain is positioned over the DNA binding
region blocking the access to DNA. A large conformational
rearrangement is therefore required for target DNA binding.
This rearrangement may also occur in TrwB, which would
provide a putative working mechanism (see below). XerD
site-specific recombination has been associated with FtsK
[105], a protein that shares functional and structural features
with TrwB and other T4CPs (see above). XerD works in
conjugation with FtsK to resolve the knots formed after
replication of the bacterial chromosome. If TrwB AAD was
the functional counterpart of XerD it could be involved in
DNA binding or some form of processing. A central pore is
also shaped by α helices from the constituting subunits in
six-clawed grapple-like Cag525. These subunits do not
comprise a distinct subdomain, but they are localised on the
convex side of the central β-sheet of the NBD [138].
The TrwB all-α domain displays further topological
similarity within a ~40-residue segment encompassing three
α-helices with the recently reported solution structure of the
56-residue DNA-binding domain of TraM, a component of
the relaxosome encoded by the dtr region of plasmid R1
[140]. This protein enhances relaxase activity and it is
capable of binding DNA [141]. TraM specifically interacts
with the TrwB orthologue in IncF plasmids, TraD,
suggesting that TraM links the relaxosome with the DNA
transfer apparatus [98]. Plasmid R388 lacks such a TraM
orthologue. Therefore, an appealing hypothesis is that TrwB
has imbedded in its structure a DNA-binding domain similar
to that of TraM, that may directly recruit the relaxosome.
Nevertheless, on examining the hexameric toroidal
quaternary structures of the protein families mentioned (Figs.
(2c-f)), helicases, AAA ATPases and Cag525 appear more
flat-topped than TrwB, with much less interaction surface
between the constituting protomers. This is needed in
1558
Current Pharmaceutical Design, 2004, Vol. 10, No. 13
Gomis-Rüth et al.
Fig 2. (a) Ribbon diagram of a TrwB protomer, with its transmembrane domain tentatively modeled (gray). The constituting
domains 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 traversing
the 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 are
presented.
Coupling Factors in Macromolecular Type-IV Secretion Machineries
helicases to allow interchanging aggregation stages and
helicoidal protein-filament formation [126, 127, 142]. The
hexameric structure of TrwB, with almost 50% of the surface
of a monomer shaping the interface to vicinal protomers
almost rules out other oligomerisation states. Most striking
structural similarity of the TrwB oligomer is found to both α
and β subunits of F1-ATPase, part of the membraneassociated F0F1-ATPase complex responsible for energy
conversion through axial rotation movement in mitochondria, chloroplasts, and bacteria (PDB 1bmf; [143]), suggesting also a link on the functional level (see below). The
overall hexamer dimensions and the almost spherical shape
are much more reminiscent of the F1-ATPase α3β3 heterohexamer. Like TrwB, this protein is a membrane protein.
WORKING
T4CPS
HYPOTHESIS
FOR
CONJUGATIVE
The mechanism of action of type-IV secretion has not
been experimentally established yet for all its steps. Hereby,
we discuss alternative hypothetical functions for TrwB and
other T4CPs, that may act as a motor pushing the T-DNA to
the transport apparatus, as simple bridging proteins between
two macromolecular assemblies, the relaxosome and the
type-IV transport machinery, or as true DNA pumps that
thread the T-strand through the pore connecting the donor
and the recipient cells. This latter function could be part of a
“shot and pump” model, following which T4CPs would
catalyse active pumping of T-DNA to the recipient once the
type-IV secretion system has shuttled a pilot protein, the
relaxase and helicase (TrwC in the R388 system, see above),
with the covalently bound T-DNA through the pilus
appendage to the acceptor cell [77]. In the first and third
T4CP functional hypotheses, ATP hydrolysis, probably
triggered by interactions with other cellular components, like
the relaxosome itself, or with membrane lipids, should be
necessary. Furthermore, this hydrolytic activity might be
associated with an axial rotating motion to translocate DNA.
The first model portrays TrwB as a rotary machine. The
TrwB hexamer displays an exterior surface belt with highly
positive charges that may explain the capability of the
protein to bind ss and dsDNA non-specifically [82]. Accordingly, ssDNA, after being processed by the nicking enzyme
and helicase TrwC, may wrap around a TrwB hexamer (Fig.
(3a)). The hexamer could behave as a membrane-anchored
motor spinning around its vertical axis (following the F0F1ATPase analogy). Relaxosome T-DNA would be translocated by TrwB to the mating apparatus and there cross the pore
to the recipient cell. Electron microscopy images of
elongated aggregates of TrwB molecules and DNA [82] may
support this hypothesis, but there is no further evidence for
this wrapping model. Mutation studies concerning basic
residues of the belt show no functional implications.
Therefore, this model is probably the less likely one.
The second hypothesis implies that the coupling protein
is not a pump or motor at all. It would behave just as a twosided recognition protein recruiting the relaxosome by
contacting its components on one side and the innermembrane part of the type-IV transport machine on another
side (Fig. (3b)). This model does not require an ATPase
activity, although a nucleotide-binding-induced molecular
Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1559
switch is reasonable. Furthermore, it would be difficult to
explain the presence of a nucleotide-binding site otherwise.
No direct ATPase activity has been observed for
TrwB∆N70, but this is not a concluding result, since
cleavage of the transmembrane part may have hindered the
enzyme activity. The completely transfer-deficient K136T
mutant of TrwB binds ATP normally, so deficiency must be
somehow related with hydrolysis. Moreover, some other
components - relaxosome, DNA, etc. - may be required to
trigger TrwB functionality.
The structure of H. pylori Cag525 protein [138], a
member of the VirB11/PulE family of ATPases [144]
involved in the type-IV secretion system of the latter
pathogen, reveals a six-clawed grapple shape mounted on a
hexameric ring and it is proposed to be membrane-associated. It has been shown to function as dynamic hexameric
assemblies with nucleotide-dependent conformational
changes regulating substrate export or T4SS assembly [145].
The membrane-distal surface of the hexamer is formed by a
helical bundle at the C-terminus of the RecA-like α/β
domain. The six bundles restrict the size of the central pore
to ∼10 Å (Fig. (2e)). An orthologue of this protein is also
present in the secretory organelle of plasmid R388 in the
form of TrwD [146]. Except for the RecA-like α/β core and
the hexameric nature of the protein, the TrwB and Cag525
structures strongly differ. As the latter ATPase may participate in the trafficking of 150-kDa CagA protein to gastric
or duodenal epithelial cells during pathogenesis of H. pylori,
the authors propose a mechanism of ATP-dependent opening/
closure of the pore following domain rearrangement and
subsequent protein export through this pore [138]. If the
equivalent TrwD plays such a role in the conjugation system
transporting DNA or a protein/DNA complex instead of the
CagA toxin, TrwB would probably not have the same
function. However, TrwB has a real transmembrane domain,
whereas the evidence for TrwD or Cag525 protein crossing
the membrane is more circumstantial.
Finally, ssDNA may pass through the central hexamer
channel, may be injected through the inner membrane into
the periplasmic space and there to the translocon (Fig. (3c)).
By the reasons mentioned before, this is the preferred
alternative. Thus, conjugal T4CPs would be behaving as
NTP-dependent DNA pumps that transfer the T-strand
through a type-IV system during conjugation, in a fashion
similar to SpoIIIE/FtsK-like proteins or as has been postulated for RuvB, which is capable of dsDNA translocation
[100, 101, 147]. The channel is basically of electronegative
nature, but it possesses belts of positively charged (arginine
and lysine) amino acids (Fig. (2b)). Taking into account that
the 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 a
reovirus, which is traversed by a RNA molecule [148], and
for the bacteriophage Φ29 connector particle [149, 150]. The
side of the TrwB channel facing the inner-membrane has an
opening of ∼ 22 Å, compatible with the values of 25-30 Å
for RecA-like hexameric helicases, through which ssDNA
may pass [151]. Functional T7 gene 4 ring helicase has an
even narrower central hole, of ~15 Å [130]. The main
objection to this hypothesis is the narrow entrance to the
channel (∼7-8 Å). However, constriction here may impose a
1560
Current Pharmaceutical Design, 2004, Vol. 10, No. 13
level of regulation that prevents continuous leakage of the
cell contents. On the other hand, the bacterial cell may
prevent permeation of the membrane for ions and small
molecules through this narrow though open channel. Thus,
the channel may be even further constricted at the
transmembrane part, remaining impermeable until a certain
stimulus triggers the opening, e.g. interaction with the
forming relaxosome. This may occur by domain
rearrangement, as has been proposed for the structurally
similar domain 1 of XerD (see above). Accordingly, a slight
motion around the hinge region between the all-α domain
and NBD may increase the channel diameter to permit
ssDNA threading (Fig. (3c)).
CONCLUSION
With the present data, it is still difficult to define the
precise mechanism of DNA transport during conjugation,
but, for the first time, crucial structural data on three of the
constituting parts of type-IV secretion systems and their
accessory proteins are available (TrwB, Cag525 and TraM).
The first two have been found to be toroidal hexamers. Such
multimeric ring-shaped structures are associated with distinct
activities like proteolysis, folding assistance, migration along
nucleic acids, cell-cycle regulation and proton pumping,
among others [152]. A toroidal protein oligomerisation is
characterised by the presence of a channel or pore traversing
Gomis-Rüth et al.
the particle and thus connecting two environments that may
differ or forming a cavity that harbours the catalytic site(s).
When the oligomerisation involves a single or similar
protomers, the particle contains several (potential) active
sites. Formation of a toroid via oligomerisation has the
advantage that construction of a circular structure by
multiplication and assembly of equal subunits is simpler and
requires less genetic information than the design of one large
protein with a central cavity. Moreover, it generates multiple
identical binding sites that may allow sequential binding and
release of substrates or interacting molecules without
dissociation from the ring, as suggested for ring helicases
while translocating along nucleic acids. Another advantage is
the formation of a topological link with the transport
macromolecule, avoiding dissociation and conferring
processivity, and thus achieving the speed necessary for
manipulation of large macromolecules within the short time
frame required for translocation or secretion.
ACKNOWLEDGEMENTS
This study was supported by grants BIO2000-1659,
BIO2002-00063, BIO2002-03964, and BIO2003-00132 (to
M.C and F.X.G.R) and by BMC2002-00379 (to F.C.) from
the Ministerio de Ciencia y Tecnología, Spain, and by grant
2001SGR346 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 before
transfer [112, 113]. TrwA is a small, tetrameric protein with a dual role in conjugation [114]. It binds to two sites at oriT and enhances TrwC
relaxase 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, the
relaxosome contacts the coupling protein. (a) Hypothesis suggesting that the T-strand DNA wraps around the particle. Vertical spinning
energised 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 the
central channel upon rearrangement of the all-α domains to enlarge the cytoplasmic entrance. In this case, ATP-mediated spinning or
sequential rearrangements in the channel may allow pumping of the T-strand into the periplasm.
Coupling Factors in Macromolecular Type-IV Secretion Machineries
REFERENCES
Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1561
[24]
References 210-212 are related articles recently published in
Current Pharmaceutical Design.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
Nagai H, Roy CR. Show me the substrates: modulation of host cell
function by type IV secretion systems. Cell Microbiol 2003; 5:
373-83.
Gomis-Rüth FX, de la Cruz F, Coll M. Structure and role of
coupling proteins in conjugal DNA transfer. Res Microbiol 2002;
153: 199-204.
Plano GV, Day JB, Ferracci F. Type III export: new uses for an old
pathway. Mol Microbiol 2001; 40: 284-93.
Linton KJ, Higgins CF. The Escherichia coli ATP-binding casette
(ABC) proteins. Mol Microbiol 1998; 28: 5-13.
Jacob-Dubuisson F, Locht C, Antoine R. Two-partner secretion in
Gram-negative bacteria: a thrifty, specific pathway for large
virulence proteins. Mol Microbiol 2001; 40: 306-13.
Sandqvist M. Biology of type II secretion. Mol Microbiol 2001; 40:
271-83.
Salmond GPC. Secretion of Extracellular Virulence Factors by
Plant Pathogenic Bacteria. Annu Rev Phytopathol 1994; 32: 181200.
Krause S, Pansegrau W, Lurz R, de la Cruz F, Lanka E.
Enzymology of type IV macromolecule secretion systems: the
conjugative transfer regions of plasmids RP4 and R388 and the cag
pathogenicity island of Helicobacter pylori encode structurally and
functionally related nucleoside triphosphate hydrolases. J Bacteriol
2000; 182: 2761-70.
Burns DL. Biochemistry of type IV secretion. Curr Op Microbiol
1999; 2: 25-9.
Galán JE, Collmer A. Type III secretion machines: bacterial
devices for protein delivery into host cells. Science 1999; 284:
1322-8.
Cao TB, Saier Jr MH. Conjugal type IV macromolecular transfer
systems of Gram-negative bacteria: organismal distribution,
structural constraints and evolutionary conclusions. Microbiology
2001; 147: 3201-14.
Saier Jr MH. A functional-phylogenetic classification system for
transmembrane solute transporters. Microbiol Mol Biol Rev 2000;
64: 354-411.
Kostelidou K, Thomas CM. DNA recognition by the KorA proteins
of IncP-1 plasmids RK2 and R751. Biochim Biophys Acta 2002;
1576: 110-8.
Christie PJ. Type IV secretion: intercellular transfer of
macromolecules by systems ancestrally related to conjugation
machines. Mol Microbiol 2001; 40: 294-305.
Christie PJ, Vogel JP. Bacterial type IV secretion: conjugation
systems adapted to deliver effector molecules to host cells. Trends
in Microbiol 2000; 8: 354-60.
Covacci A, Telford JL, del Giudice G, Parsonnet J, Rappuoli R.
Helicobacter pylori virulence and genetic geography. Science
1999; 284: 1328-33.
O'Callaghan D, Cazevieille C, Allardet-Servent A, Boschiroli ML,
Bourg G, Foulongne V, et al. A homologue of the Agrobacterium
tumefaciens VirB and Bordetella pertussis Ptl type IV secretion
systems is essential for intracellular survival of Brucella suis. Mol
Microbiol 1999; 33: 1210-20.
Anthony KG, Sherburne C, Sherburne R, Frost LS. The role of the
pilus in recipient cell recognition during bacterial conjugation
mediated by F-like plasmids. Molec Microbiol 1994; 13: 939-53.
Zupan JR, Ward D, Zambryski P. Assembly of the VirB transport
complex for DNA transfer from Agrobacterium tumefaciens to
plant cells. Curr Op Microbiol 1998; 1: 649-55.
Winans SC, Burns DL, Christie PJ. Adaptation of a conjugal
transfer system for the export of pathogenic macromolecules.
Trends Microbiol 1996; 4: 64-8.
Christie PJ. Agrobacterium tumefaciens T-complex transport
apparatus: a paradigm for a new family of multifunctional
transporters in eubacteria. J Bacteriol 1997; 179: 3085-94.
Frost LS, Ippen-Ihler K, Skurray RA. Analysis of the sequence and
gene products of the transfer region of the F sex factor. Microbiol
Rev 1994; 58: 162-210.
Piper KR, Farrand SK. Quorum sensing but not autoinduction of Ti
plasmid conjugal transfer requires control by the opine regulon and
the antiactivator TraM. J Bacteriol 2000; 182: 1080-8.
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
Firth N, Ippen-Ihler K, Skurray RA. In: Neidhart FC, Curtiss III R,
Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS,
Riley M, Schaechter M, Umbarger HC Eds, Escherichia coli and
Salmonella: cellular and molecular biology. Washington, D.C.,
American Society for Microbiology 1996; 2377-401.
Buchanan-Wollaston V, Passiatore JE, Cannon F. The mob and
oriT mobilization functions of a bacterial plasmid promote its
transfer to plants. Nature 1987; 328: 172-5.
Zechner EL, de la Cruz F, Eisenbrandt R, Grahn AM, Koraimann
G, Lanka E, et al. In: Thomas CM Eds, The horizontal gene pool:
bacterial plasmids and gene spread. London, Harwood Academic
Publishers 2000; 87-173.
Lanka E, Wilkins BM. DNA processing reactions in bacterial
conjugation. Annu Rev Biochem 1995; 64: 141-69.
Heinemann JA. Genetics of gene transfer between species. Trends
in Genetics 1991; 7: 181-5.
Heinemann JA, Sprague GFJ. Bacterial conjugative plasmids
mobilize DNA transfer between bacteria and yeast. Nature 1989;
340: 205-9.
Lessl M, Lanka E. Common mechanisms in bacterial conjugation
and Ti-mediated T-DNA transfer to plant cells. Cell 1994; 77: 3214.
Grahn AM, Haase J, Bamford D, Lanka E. Components of the RP4
conjugative transfer apparatus from an envelope structure bridging
inner and outer membranes of donor cells: implications for related
macromolecule transport systems. J Bacteriol 2000; 182: 1564-74.
Lawley TD, Klimke WA, Gubbins MJ, Frost LS. F factor
conjugation is a true type IV secretion system. FEMS Microbiol
Lett 2003; 224: 1-15.
Beijersbergen A, den Dulk-Ras A, Schilperoort RA, Hooykaas PJJ.
Conjugative transfer by the virulence system of Agrobacterium
tumefaciens. Science 1992; 256: 1324-7.
Heinemann JA. Genetic evidence of protein transfer during
bacterial conjugation. Plasmid 1999; 41: 240-7.
Vergunst AC, Schrammeijer B, den Dulk-Ras A, de Vlaam CMT,
Regensburg-Tuïnk TJG, Hooykaas PJJ. VirB/D4-dependent protein
translocation from Agrobacterium into plant cells. Science 2000;
290: 979-82.
Li P-L, Hwang I, Miyagi H, True H, Farrand SK. Essential
components of the Ti plasmid trb system, a type IV
macromolecular transporter. J Bacteriol 1999; 181: 5033-41.
de la Cruz F, Lanka E. In: Spaink HP, Kondorosi A, Hooykaas PJJ
Eds, The rhizobiaceae. Dordrecht, Netherland, Kluwer Academic
Publishing 1998; 281-301.
Tzfira T, Rhee Y, MHC, Kunik T, Citovsky V. Nucleic acid
transport in plant-microbe interactions: the molecules that walk
through the walls. Annu Rev Microbiol 2000; 54: 187-219.
D'Halluin K, Botterman J. In: Spaink HP, Kondorosi A, Hooykaas
PJJ Eds, The rhizobiaceae: molecular biology of model plantassociated bacteria. Amsterdam, Kluwer Academic Publishers
1998; 339-45.
Lai E-M, Kado CI. The T-pilus of Agrobacterium tumefaciens.
Trends in Microbiol 2000; 8: 361-9.
de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen A.
Agrobacterium
tumefaciens-mediated
transformation
of
filamentous fungi. Nat Biotech 1998; 16: 839-42.
Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V.
Genetic transformation of HeLa cells by Agrobacterium. Proc Natl
Acad Sci USA 2001; 98: 1871-6.
Chen L, Chen Y, Wood DW, Nester EW. A new type IV secretion
system promotes conjugal transfer in Agrobacterium tumefaciens. J
Bacteriol 2002; 184: 4838-45.
Leloup L, Lai EM, Kado CI. Identification of a chromosomal tralike region in Agrobacterium tumefaciens. Mol Genet Genomics
2002; 267: 115-23.
Smith LD, Ficht TA. Pathogenesis of Brucella. Crit Rev Microbiol
1990; 17: 209-30.
Shuman HA, Horwitz MA. Legionella pneumophila invasion of
mononuclear phagocytes. Curr Top Microbiol Immunol 1996; 209:
99-112.
Ohashi N, Zhi N, Lin Q, Rikihisa Y. Characterization and
transcriptional analysis of gene clusters for a type IV secretion
machinery in human granulocytic and monocytic ehrlichiosis
agents. Infect Immun 2002; 70: 2128-38.
Sieira R, Comerci DJ, Sánchez DO, Ugalde RA. A homologue of
an operon required for DNA transfer in Agrobacterium is required
1562
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
Current Pharmaceutical Design, 2004, Vol. 10, No. 13
in Brucella abortus for virulence and intracellular multiplication. J
Bacteriol 2000; 182: 4849-55.
Hong PC, Tsolis RM, Ficht TA. Identification of genes required for
chronic persistence of Brucella abortus in mice. Infect Immun
2000; 68: 4102-7.
Boschiroli ML, Ouahrani-Bettache S, Foulongne V, MichauxCharachon S, Bourg G, Allardet-Servent A, et al. The Brucella suis
virB operon is induced intracellularly in macrophages. Proc Natl
Acad Sci USA 2002; 99: 1544-9.
del Vecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, Los T,
et al. The genome sequence of the facultative intracellular pathogen
Brucella melitensis. Proc Natl Acad Sci USA 2002; 99: 443-8.
Delrue RM, Martínez-Lorenzo M, Lestrate P, Danese I, Bielarz V,
Mertens P, et al. Identification of Brucella spp. genes involved in
intracellular trafficking. Cell Microbiol 2001; 3: 487-97.
Andersson SGE, Zomorodipour A, Andersson JO, Sicheritz-Ponten
T, Alsmark UCM, Podowski RM, et al. The genome sequence of
Rickettsia prowazekii and the origin of mitochondria. Nature 1998;
396: 133-43.
Segal G, Shuman HA. How is the intracellular fate of the
Legionella pneumophila phagosome determined? Trends in
Microbiol 1998; 6: 253-5.
Cianciotto NP. Pathogenicity of Legionella pneumophila. Int J Med
Microbiol 2001; 291: 331-43.
Hobbs M, Mattick JS. Common components in the assembly of
type 4 fimbriae, DNA transfer systems, filamentous phage and
protein-secretion apparatus: a general system for the formation of
surface-associated protein complexes. Mol Microbiol 1993; 10:
233-43.
Zink SD, Pedersen L, Cianciotto NP, Abu-Kwaik Y. The Dot/Icm
type IV secretion system of Legionella pneumophila is essential for
the induction of apoptosis in human macrophages. Infect Immun
2002; 70: 1657-63.
Nagai H, Roy CR. The DotA protein from Legionella pneumophila
is secreted by a novel process that requires the Dot/Icm transporter.
EMBO J 2001; 20: 5962-70.
Vogel JP, Andrews HL, Wong SK, Isberg RR. Conjugative transfer
by the virulence system of Legionella pneumophila. Science 1998;
279: 873-6.
Segal G, Purcell M, Shuman HA. Host cell killing and bacterial
conjugation require overlapping sets of genes within a 22-kb region
of the Legionella pneumophila genome. Proc Natl Acad Sci USA
1998; 95: 1669-74.
Segal G, Russo JJ, Shuman HA. Relationships between a new type
IV secretion system and the icm/dot virulence system of Legionella
pneumophila. Mol Microbiol 1999; 34: 709-809.
Felek S, Huang H, Rikihisa Y. Sequence and expression analysis of
virB9 of the type IV secretion system of Ehrlichia canis strains in
ticks, dogs, and cultured cells. Infect Immun 2003; 71: 6063-7.
Economou A. Following the leader: bacterial protein export
through the Sec pathway. Trends Microbiol 1999; 7: 315-20.
Thanassi DG, Hultgren SJ. Multiple pathways allow protein
secretion across the bacterial outer membrane. Curr Opin Cell Biol
2000; 12: 420-30.
Cheng KS, Lu MC, Tang HL, Chou FT. Phosphorylation of
Helicobacter pylori CagA in patients with gastric ulcer and
gastritis. Adv Ther 2002; 19: 85-90.
Marshall B. Helicobacter pylori: 20 years on. Clin Med 2002; 2:
147-52.
Christie PJ. The cag pathogenicity island: mechanistic insights.
Trends in Microbiol 1997; 5: 264-5.
Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W, Haas R.
Translocation of Helicobacter pylori CagA into gastric epithelial
cells by type IV secretion. Science 2000; 287: 1497-500.
Backert S, Churin Y, Meyer TF, Helicobacter pylori type IV
secretion, host cell signalling and vaccine development. Keio J
Med 2002; 51 (Suppl. 2): 6-14.
Kuipers EJ, Israel DA, Kusters JG, Blaser MJ. Evidence for a
conjugation-like mechanism of DNA transfer in Helicobacter
pylori. J Bacteriol 1998; 180: 2901-5.
Smeets LC, Kusters JG. Natural transformation in Helicobacter
pylori: DNA transport in an unexpected way. Trends Microbiol
2002; 10: 159-62.
Hofreuter D, Odenbreit S, Haas R. Natural transformation
competence in Helicobacter pylori is mediated by the basic
Gomis-Rüth et al.
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
[91]
[92]
[93]
[94]
components of a type IV secretion system. Mol Microbiol 2001;
41: 379-91.
Suerbaum S, Josenhans C, Sterzenbach T, Drescher B, Brandt P,
Bell M, et al. The complete genome sequence of the carcinogenic
bacterium Helicobacter hepaticus. Proc Natl Acad Sci USA 2003;
100: 7901-6.
Strauch E, Goelz G, Knabner D, Konietzny A, Lanka E, Appel B.
A cryptic plasmid of Yersinia enterocolitica encodes a conjugative
transfer system related to the regions of CloDF13 Mob and IncX
Pil. Microbiology 2003; 149: 2829-45.
Weiss AA, Johnson FD, Burns DL. Molecular characterization of
an operon required for pertussis toxin secretion. Proc Natl Acad Sci
USA 1993; 90: 2970-4.
Cabezón E, Sastre JI, de la Cruz F. Genetic evidence of a coupling
role for the TraG protein family in bacterial conjugation. Mol Gen
Genet 1997; 254: 400-6.
Llosa M, Gomis-Rüth FX, Coll M, de la Cruz F. Bacterial
conjugation: a two-step mechanism for DNA transport. Mol
Microbiol 2002; 45: 1-8.
Selbach M, Moese S, Meyer TF, Backert S. Functional analysis of
the Helicobacter pylori cag pathogenicity island reveals both
VirD4-CagA-dependent
and
VirD4-CagA-independent
mechanisms. Infect Immun 2002; 70: 665-71.
Kumar RB, Das A. Polar location and functional domains of the
Agrobacterium tumefaciens DNA transfer protein VirD4. Mol
Microbiol 2002; 43: 1523-32.
Okamoto S, Toyoda-Yamamoto A, Ito K, Takebe I, Machida Y.
Localization and orientation of the VirD4 protein of Agrobacterium
tumefaciens in the cell membrane. Mol Gen Genet 1991; 228: 2432.
Panicker MM, Minkley EG. Purification and properties of the F sex
factor TraD protein, an inner membrane conjugal transfer protein. J
Biol Chem 1992; 267: 12761-6.
Moncalián G, Cabezón E, Alkorta I, Valle M, Moro F, Valpuesta
JM, et al. Characterization of ATP and DNA binding activities of
TrwB, the coupling protein essential in plasmid R388 conjugation.
J Biol Chem 1999; 274: 36117-24.
Balzer D, Pansegrau W, Lanka E. Essential motifs of relaxase
(TraI) and TraG proteins involved in conjugative transfer of
plasmid RP4. J Bacteriol 1994; 176: 4285-95.
Lessl M, Pansegrau W, Lanka E. Relationship of DNA-transfer
systems: essential transfer factors of plasmids RP4, Ti and F share
common sequences. Nucl Acids Res 1992; 20: 6099-100.
Llosa M, Bolland S, de la Cruz F. Genetic organization of the
conjugal DNA processing region of the IncW plasmid R388. J Mol
Biol 1994; 235: 448-64.
Das A, Xie Y-H. Construction of transposon Tn3phoA: its
application in defining the membrane topology of the
Agrobacterium tumefaciens DNA transfer proteins. Mol Microbiol
1998; 27: 405-14.
Lee MH, Kosuk N, Bailey J, Traxler B, Manoil C. Analysis of F
factor TraD membrane topology by use of gene fusions and
trypsin-sensitive insertions. J Bacteriol 1999; 181: 6108-13.
Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly related
sequences in the α- and β-subunits of ATP synthase, myosin,
kinases and other ATP-requiring enzymes and a common
nucleotide binding fold. EMBO J 1982; 1: 945-51.
Santini JM, Stanisch VA. Both the fipA gene of pKM101 and the
pifC gene of F inhibit conjugal transfer of RP1 by an effect on
traG. J Bacteriol 1998; 180: 4093-101.
Berger BR, Christie PJ. Genetic complementation analysis of the
Agrobacterium tumefaciens virB operon: virB2 through virB11 are
essential virulence genes. J Bacteriol 1994; 176: 3646-60.
Lessl M, Balzer D, Pansegrau W, Lanka E. Sequence similarities
between the RP4 Tra2 and the Ti VirB region strongly support the
conjugation model for T-DNA transfer. J Biol Chem 1992; 267:
20471-80.
Szpirer CY, Faelen M, Couturier M. Interaction between the RP4
coupling protein TraG and the pBHR1 mobilization protein Mob.
Mol Microbiol 2000; 37: 1283-92.
Cabezón E, Lanka E, de la Cruz F. Requirements for mobilization
of plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwB
and RP4 traG are interchangeable. J Bacteriol 1994; 176: 4455-8.
Hamilton CM, Lee H, Li P-L, Cook DM, Piper KR, Beck von
Bodman S, et al. TraG from RP4 and TraG and VirD4 from Ti
Coupling Factors in Macromolecular Type-IV Secretion Machineries
[95]
[96]
[97]
[98]
[99]
[100]
[101]
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
[117]
[118]
plasmids confer relaxosome specificity to the conjugal transfer
system of pTiC58. J Bacteriol 2000; 182: 1541-8.
Sastre JI, Cabezon E, de la Cruz F. The carboxyl terminus of
protein TraD adds specificity and efficiency to F-plasmid
conjugative transfer. J Bacteriol 1998; 180: 6039-42.
Karplus K. Hidden Markov models for detecting remote protein
homologies. Bioinformatics 1998; 14: 846-56.
Schröder G, Krause S, Zechner EL, Traxler B, Yeo HJ, Lurz R,
et al. TraG-like proteins of DNA transfer systems and of the
Helicobacter pylori type IV secretion system: inner membrane gate
for exported substrates? J Bacteriol 2002; 184: 2767-79.
Disque-Kochem C, Dreiseikelmann B. The cytoplasmic DNAbinding protein TraM binds to the inner membrane protein TraD in
vitro. J Bacteriol 1997; 179(19): 6133-7.
Gilmour MW, Gunton JE, Lawley TD, Taylor DE. Interaction
between the IncHI1 plasmid R27 coupling protein and type IV
secretion system: TraG associates with the coiled-coil mating pair
formation protein TrhB. Mol Microbiol 2003; 49: 105-16.
Bath J, Wu LJ, Errington J, Wang JC. Role of Bacillus subtilis
SpoIIIE in DNA transport across the mother cell-prespore division
sepyum. Science 2000; 290: 995-7.
Wu LJ, Lewis PJ, Allmansberger R, Hauser PM, Errington J. A
conjugation-like mechanism for prespore chromosome partitioning
during sporulation in bacillus subtilis. Gen Dev 1995; 9: 1316-26.
Errington J, Bath J, Wu LJ. DNA transport in bacteria. Nat Rev
Mol Cell Biol 2001; 2: 538-45.
Sharp MD, Pogliano K. Role of cell-specific SpoIIIE assembly in
polarity of DNA transfer. Science 2002; 295: 137-9.
Wu LJ, Errington J. Septal localization of the SpoIIIE chromosome
partitioning protein in Bacillus subtilis. EMBO J 1997; 16: 2161-9.
Recchia GD, Aroyo M, Wolf D, Blakely G, Sherratt DJ. FtsKdependent and -independent pathways of Xer site-specific
recombination. EMBO J 1999; 18: 5724-34.
Steiner W, Liu GW, Donachie WD, Kuempel P. The cytoplasmic
domain of FtsK protein is required for resolution of chromosome
dimers. Mol Microbiol 1999; 31: 579-83.
Datta N, Hedges RW. Trimethoprim resistance conferred by W
plasmids in Enterobacteriaceae. J Gen Microbiol 1972; 72: 349-55.
Swift G, McCarthy BJ, Heffron F. DNA sequence of plasmidencoded dihydrofolat reductase. Mol Gen Genet 1981; 181: 441-7.
Swedberg G, Skold O. Plasmid-borne sulfonamide resistance
determinants studied by restriction enzyme analysis. J Bacteriol
1983; 153: 1228-37.
Bolland S, Llosa M, Avila P, de la Cruz F. General organization of
the conjugal transfer genes of the IncW plasmid R388 and
interactions between R388 and IncN and IncP plasmids. J Bacteriol
1990; 172: 5795-802.
Guasch A, Lucas M, Cabezas M, Pérez-Luque R, Gomis-Rüth FX,
de la Cruz F, et al. Recognition and processing of the origin of
transfer DNA by conjugative relaxase TrwC. Nat Struct Biol 2003;
10, 1002-1010.
Llosa M, Grandoso G, de la Cruz F. Nicking activity of TrwC
directed against the origin of transfer of the IncW plasmid R388. J
Mol Biol 1995; 246: 54-62.
Grandoso G, Llosa M, Zabala JC, de la Cruz F. Purification and
biochemical characterization of TrwC, the helicase involved in
plasmid R388 conjugal DNA transfer. Eur J Biochem 1994; 226:
403-12.
Moncalián G, Grandoso G, Llosa M, de la Cruz F. oriT-processing
and regulatory roles of TrwA protein in plasmid R388 conjugation.
J Mol Biol 1997; 270: 188-200.
Rice PA, Yang S-W, Mizuuchi K, Nash HA. Crystal structure of an
IHF-DNA complex: a protein induced DNA U-turn. Cell 1996; 87:
1295-306.
Moncalián G, Valle M, Valpuesta JM, de la Cruz F. IHF protein
inhibits cleavage but not assembly of plasmid R388 relaxosomes.
Mol Microbiol 1999; 31: 1643-52.
Gomis-Rüth FX, Moncalián G, Pérez-Luque R, González A,
Cabezón E, de la Cruz F, et al. The bacterial conjugation protein
TrwB resembles ring helicases and F1-ATPase. Nature 2001; 409:
637-41.
Gomis-Rüth FX, Moncalián G, de la Cruz F, Coll M. Conjugative
plasmid protein TrwB, an integral membrane type IV secretion
system coupling protein: detailed structural features and mapping
of the active-site cleft. J Biol Chem 2002; 277: 7556-66.
Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1563
[119]
[120]
[121]
[122]
[123]
[124]
[125]
[126]
[127]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
[135]
[136]
[137]
[138]
[139]
[140]
[141]
[142]
[143]
[144]
[145]
Dalal S, Hanson PI. Membrane traffic: what drives the AAA
motor? Cell 2001; 104: 5-8.
Vale RD. AAA proteins: lords of the ring. J Cell Biol 2000; 150:
F13-F9.
Guenther B, Onrust R, Sali A, O'Donnell M, Kuriyan J. Crystal
structure of the δ' subunit of the clamp-loader complex of E.coli
DNA polymerase III. Cell 1997; 91: 335-45.
Lenzen CU, Steinmann D, Whiteheart SW, Weis WI. Crystal
structure of the hexamerization domain of N-ethylmaleimidesensitive fusion protein. Cell 1998; 94: 525-35.
Zhang X, Shaw A, Bates PA, Newman RH, Gowen B, Orlova E,
et al. Structure of the AAA ATPase p97. Mol Cell 2000; 6: 147384.
Bochtler M, Hartmann C, Song HK, Bourenkov GP, Bartunik HD,
Huber R. The structures of HsIU and the ATP-dependent protease
HsIU-HsIV. Nature 2000; 403: 800-5.
Waksman G, Lanka E, Carazo J-M. Helicases as nucleic acid
unwinding machines. Nat Struct Biol 2000; 7: 20-2.
Soultanas P, Wigley DB. DNA helicases: 'inching forward'. Curr
Op Struct Biol 2000; 10: 124-8.
Sawaya MR, Guo S, Tabor S, Richardson CC, Ellenberger T.
Crystal structure of the helicase domain from the replicative
helicase-primase of bacteriophage T7. Cell 1999; 99: 167-77.
Hacker KJ, Johnson KA. A hexameric helicase encircles one DNA
strand and excludes the other during DNA unwinding.
Biochemistry 1997; 36: 14080-7.
Schwer B. A new twist on RNA helicases: DExxH/D box proteins
as RNPases. Nat Struct Biol 2001; 8: 113-6.
Singleton MR, Sawaya MR, Ellenberger T, Wigley DB. Crystal
structure of T7 gene 4 ring helicase indicates a mechanism for
sequential hydrolysis of nucleotides. Cell 2000; 101: 589-600.
Niedenzu T, Röleke D, Bains G, Scherzinger E, Saenger W.
Crystal structure of the hexameric replicative helicase RepA of
plasmid RSF1010. J Mol Biol 2001; 306: 479-87.
Yu X, Egelman EH. The RecA hexamer is a structural homologue
of ring helicases. Nat Struct Biol 1997; 4(2): 101-4.
Story RM, Weber IT, Steitz TA. The structure of the E.coli recA
protein monomer and polymer. Nature 1992; 355: 318-25.
Story RM, Steitz TA. Structure of the recA protein - ADP complex.
Nature 1992; 355: 374-6.
Subramanya HS, Bird LE, Brannigan JA, Wigley DB. Crystal
structure of a DExx box DNA helicase. Nature 1996; 384: 379-83.
Velankar SS, Soultanas P, Dillingham MS, Subramanya HS,
Wigley DB. Crystal structures of complexes of PcrA DNA helicase
with a DNA substrate indicate an inchworm mechanism. Cell 1999;
97: 75-84.
Machón C, Rivas S, Albert A, Goñi FM, de la Cruz F. TrwD, the
hexameric traffic ATPase encoded by plasmid R388, induces
membrane destabilization and hemifusion of lipid vesicles. J
Bacteriol 2002; 184: 1661-8.
Yeo H-J, Savvides SN, Herr AB, Lanka E, Waksman G. Crystal
structure of the hexameric traffic ATPase of the Helicobacter
pylori type IV secretion system. Mol Cell 2000; 6: 1461-72.
Subramanya HS, Arciszewska LK, Baker RA, Bird LE, Sherratt
DJ, Wigley DB. Crystal structure of the site-specific recombinase,
XerD. EMBO J 1997; 16: 5178-87.
Stockner T, Plugariu C, Koraimann G, Högenauer G, Bermel W,
Prytulla S, et al. Solution structure of the DNA-binding domain of
TraM. Biochemistry 2001; 40: 3370-7.
Kupelwieser G, Schwab M, Hogenauer G, Koraimann G, Zechner
EL. Transfer protein TraM stimulates TraI-catalyzed cleavage of
the transfer origin of plasmid R1 in vivo. J Mol Biol 1998; 275:
81-94.
Yu X, Shibata T, Egelman EH. Identification of a defined epitope
on the surface of the active RecA-DNA filament using a
monoclonal antibody and three-dimensional reconstruction. J Mol
Biol 1998; 283: 985-92.
Abrahams JP, Leslie AGW, Lutter R, Walker JE. Structure at 2.8 Å
resolution of F1-ATPase from bovine heart mitochondria. Nature
1994; 370: 621-8.
Montallebi-Veshareh M, Balzer D, Lanka E, Jagura-Burdzy G,
Thomas CM. Conjugative transfer functions of broad-host-range
plasmid RK2 are coregulated with vegetative replication. Mol
Microbiol 1992; 6: 907-20.
Savvides SN, Yeo HJ, Beck MR, Blaesing F, Lurz R, Lanka E,
et al. VirB11 ATPases are dynamic hexameric assemblies: new
1564
[146]
[147]
[148]
[149]
[150]
[151]
[152]
[153]
[154]
[155]
[156]
[157]
[158]
[159]
[160]
[161]
[162]
[163]
[164]
[165]
[166]
[167]
[168]
Current Pharmaceutical Design, 2004, Vol. 10, No. 13
insights into bacterial type IV secretion. EMBO J 2003; 22: 196980.
Rivas S, Bolland S, Cabezón E, Goñi FM, de la Cruz F. TrwD, a
protein encoded by the IncW plasmid R388, displays an ATP
hydrolase activity essential for bacterial conjugation. J Biol Chem
1997; 272: 25583-90.
Parsons CA, Stasiak A, Bennett RJ, West SC. Structure of a
multisubunit complex that promotes DNA branch migration.
Nature 1995; 374: 375-8.
Reinisch KM, Nibert ML, Harrison SC. Structure of the reovirus
core at 3.6 Å resolution. Nature 2000; 404: 960-7.
Simpson AA, Tao Y, Leiman PG, Badasso MO, He Y, Jardine PJ,
et al. Structure of the bacteriophage Φ29 DNA packaging motor.
Nature 2000; 408: 745-50.
Guasch A, Pous J, Ibarra B, Gomis-Rüth FX, Valpuesta JM, Sousa
N, et al. Detailed architecture of a DNA translocating machine: the
high-resolution structure of the bacteriophage Φ29 connector
particle. J Mol Biol 2002; 315(4): 663-76.
Egelman EH, Yu X, Wild R, Hingorani MM, Patel SM.
Bacteriophage T7 helicase/primase proteins form rings around
single-strandd DNA that suggest a general structure for hexameric
helicases. Proc Natl Acad Sci USA 1995; 92: 3869-73.
Hingorani MM, O'Donnell M. Toroidal proteins: Running rings
around DNA. Curr Biol 1998; 8: R83-R6.
Sullivan JT, Trzebiatowski JR, Cruickshank RW, Gouzy J, Brown
SD, Elliot RM, et al. Comparative sequence analysis of the
symbiosis island of Mesorhizobium loti strain R7A. J Bacteriol
2002; 184: 3086-95.
Bittinger MA, Gross JA, Widom J, Clardy J, Handelsman J.
Rhizobium etli CE3 carries vir gene homologs on a selftransmissible plasmid. Mol Plant Microbe Interact 2000; 13: 101921.
Segal G, Shuman HA. Possible origin of the Legionella
pneumophila virulence genes and their relation to Coxiella burnetii.
Mol Microbiol 1999; 33: 669-70.
Padmalayam I, Karem K, Baumstark B, Massung R. The gene
encoding the 17-kDa antigen in Bartonella henselae is located
within a cluster of genes homologous to the virB virulence operon.
DNA Cell Biol 2000; 19: 377-82.
Baron C, O'Callaghan D, Lanka E. Bacterial secrets of secretion:
EuroConference on the biology of type IV secretion processes. Mol
Microbiol 2002; 43: 1359-65.
Seubert A, Schulein R, Dehio C. Bacterial persistence within
erythrocytes: a unique pathogenic strategy of Bartonella spp. Int J
Med Microbiol 2002; 291: 555-60.
Seubert A, Hiestand R, de la CF, Dehio C. A bacterial conjugation
machinery recruited for pathogenesis. Mol Microbiol 2003; 49:
1253-66.
Schmiederer M, Arcenas R, Widen R, Valkov N, Anderson B.
Intracellular induction of the Bartonella henselae virB operon by
human endothelial cells. Infect Immun 2001; 69: 6495-502.
Bacon DJ, Alm RA, Burr DH, Hu L, Kopecko DJ, Ewing CP, et al.
Involvement of a plasmid in virulence of Campylobacter jejuni 81176. Infect Immun 2000; 68: 4384-90.
Bacon DJ, Alm RA, Hu L, Hickey TE, Ewing CP, Batchelor RA,
et al. DNA sequence and mutational analyses of the pVir plasmid
of Campylobacter jejuni 81-176. Infect Immun 2002; 70: 6242-50.
Novak KF, Dougherty B, Peláez M. Actinobacillus actinomycetemcomitans harbours type IV secretion system genes on a plasmid and
in the chromosome. Microbiology 2001; 147: 3027-35.
Henderson B, Nair SP, Ward JM, Wilson M. Molecular
pathogenicity of the oral opportunistic pathogen Actinobacillus
actinomycetemcomitans. Annu Rev Microbiol 2003; 57: 29-55.
Sexton JA, Vogel JP. Type IVB secretion by intracellular
pathogens. Traffic 2002; 3: 178-85.
Zamboni DS, McGrath S, Rabinovitch M, Roy CR. Coxiella
burnetii express type IV secretion system proteins that function
similarly to components of the Legionella pneumophila Dot/Icm
system. Mol Microbiol 2003; 49: 965-76.
Stouthamer R, Breeuwer JAJ, Hurst GDD. Wolbachia pipientis:
microbial manipulator of arthropod reproduction. Annu Rev
Microbiol 1999; 53: 71-102.
Masui S, Sasaki T, Ishikawa H. Genes for the type IV secretion
system in an intracellular symbiont, Wolbachia, a causative agent
of various sexual alterations in arthropodes. J Bacteriol 2000; 182:
6529-31.
Gomis-Rüth et al.
[169]
[170]
[171]
[172]
[173]
[174]
[175]
[176]
[177]
[178]
[179]
[180]
[181]
[182]
[183]
[184]
[185]
[186]
[187]
[188]
[189]
[190]
Liu H, Kang Y, Genin S, Schell MA, Denny TP. Twitching
motility of Ralstonia solanacearum requires a type IV pilus system.
Microbiology 2001; 147: 3215-29.
Jones CS, Osborne DJ. Identification of contemporary plasmid
virulence genes in ancestral isolates of Salmonella enteritidis and
Salmonella typhimurium. FEMS Microbiol Lett 1991; 64: 7-11.
da Silva ACR, Ferro JA, Reinach FC, Farah CS, Furlan LR,
Quaggio RB, et al. Comparison of the genomes of two
Xanthomonas pathogens with differing host specificities. Nature
2002; 417: 459-63.
Yoshioka Y, Fujita Y, Ohtsubo E. Nucleotide sequence of the
promoter-distal region of the tra operon of plasmid R100, including
traI (DNA helicase I) and traD genes. J Mol Biol 1990; 214: 39-53.
Jalajakumari MB, Manning PA. Nucleotide sequence of the traD
region in the Escherichia coli F sex factor. Gene 1989; 81: 195202.
Romine MF, Stillwell LC, Wong KK, Thurston SJ, Sisk EC,
Sensen C, et al. Complete sequence of a 184-kilobase catabolic
plasmid from Sphingomonas aromaticivorans F199. J Bacteriol
1999; 181: 1585-602.
Coupland GM, Brown AM, Willetts NS. The origin of transfer
(oriT) of the conjugative plasmid R46: characterization by deletion
analysis and DNA sequencing. Mol Gen Genet 1987; 208: 219-25.
Paterson ES, Moré MI, Pillay G, Cellini C, Woodgate R, Walker
GC, et al. Genetic analysis of the mobilization and leading regions
of the IncN plasmids pKM101 and pCU1. J Bacteriol 1999; 181:
2572-83.
Hirayama T, Muranaka T, Ohkawa H, Oka A. Organization and
characterization of the virCD genes from Agrobacterium
rhizogenes. Mol Gen Genet 1988; 213: 229-37.
Porter SG, Yanofsky MF, Nester EW. Molecular characterization
of the virD operon from Agrobacterium tumefaciens. Nucleic Acids
Res 1987; 15: 7503-17.
Rogowsky PM, Powell BS, Shirasu K, Lin TS, Morel P, Zyprian
EM, et al. Molecular characterization of the vir regulon of
Agrobacterium tumefaciens: complete nucleotide sequence and
gene organization of the 28.63-kbp regulon cloned as a single unit.
Plasmid 1990; 23: 85-106.
Ogata H, Audic S, Renesto-Audiffren P, Fournier P-E, Barbe V,
Samson D, et al. Mechanisms of evolution in Rickettsia conorii and
R. prowazekii. Science 2001; 293: 2093-8.
Ziegelin G, Pansegrau W, Strack B, Balzer D, Kröger M, Kruft V,
et al. Nucleotide sequence and organization of genes flanking the
transfer origin of promiscuous plasmid RP4. DNA Sequence 1991;
1: 303-27.
Komano T, Kubo A, Nisioka T. Shufflon: multi-inversion of four
contiguous DNA segments of plasmid R64 creates seven different
open reading frames. Nucleic Acids Res 1987; 15: 1165-72.
Tomb J-F, White O, Kerlavage AR, Clayton RA, Sutton GG,
Fleischmann RD, et al. The complete genome sequence of the
gastric pathogen Helicobacter pylori. Nature 1997; 388: 539-47.
Simpson AJG, Reinach FC, Arruda P, Abreu FA, Acencio M,
Alvarenga R, et al. The genome sequence of the plant pathogen
Xylella fastidiosa. Nature 2000; 406: 151-157.
Galli DM, Chen J, Novak KF, Leblanc DJ. Nucleotide sequence
and analysis of conjugative plasmid pVT745. J Bacteriol 2001;
183: 1585-94.
Freiberg C, Fellay R, Bairoch A, Broughton WJ, Rosenthal A,
Perret X. Molecular basis of symbiosis between Rhizobium and
legumes. Nature 1997; 387: 394-401.
Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S,
et al. Complete genome structure of the nitrogen-fixing symbiotic
bacterium Mesorhizobium loti. DNA Res 2000; 7: 331-8.
Alt-Morbe J, Stryker JL, Fuqua C, Li PL, Farrand SK, Winans SC.
The conjugal transfer system of Agrobacterium tumefaciens
octopine-type Ti plasmids is closely related to the transfer system
of an IncP plasmid and distantly related to Ti plasmid vir genes. J
Bacteriol 1996; 178: 4248.
Farrand SK, Hwang I, Cook DM. The tra region of the nopalinetype Ti plasmid is a chimera with elements related to the transfer
systems of RSF1010, RP4, and F. J Bacteriol 1996; 178: 4233-47.
Salanoubat M, Genin S, Artiguenave F, Gouzy J, Mangenot S,
Arlat M, et al. Genome sequence of the plant pathogen Ralstonia
solanacearum. Nature 2002; 415: 497-502.
Coupling Factors in Macromolecular Type-IV Secretion Machineries
[191]
[192]
[193]
[194]
[195]
[196]
[197]
[198]
[199]
[200]
[201]
Alarcón-Chaídez F, Sampath J, Srinivas P, Vijayakumar MN.
Tn5252: a model for complex streptococcal conjugative
transposons. Adv Exp Med Biol 1997; 418: 1029-32.
Noelling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q,
Gibson R, et al. Genome sequence and comparative analysis of the
solvent-producing bacterium Clostridium acetobutylicum. J
Bacteriol 2001; 183: 4823-38.
Schneiker S, Keller M, Droge M, Lanka E, Puhler A, Selbitschka
W. The genetic organization and evolution of the broad host range
mercury resistance plasmid pSB102 isolated from a microbial
population residing in the rhizosphere of alfalfa. Nucl Acids Res
2001; 29: 5169-81.
Morton TM, Eaton DM, Johnston JL, Archer GL. DNA sequence
and units of transcription of the conjugative transfer gene complex
(trs) of Staphylococcus aureus plasmid pGO1. J Bacteriol 1993;
175: 4436-47.
Dougherty BA, Hill C, Weidman JF, Richardson DR, Venter JC,
Ross RP. Sequence and analysis of the 60 kb conjugative
bacteriocin-producing plasmid pMRC01 from Lactococcus lactis
DPC3147. Mol Microbiol 1998; 29: 1029-38.
Garnier F, Taourit S, Glaser P, Courvalin P, Galimand M.
Characterization of transposon Tn1549, conferring VanB-type
resistance in Enterococcus spp.. Microbiology 2000; 146: 1481-9.
Nuñez B, Ávila P, de la Cruz F. Genes involved in conjugative
DNA processing of plasmid R6K. Mol Microbiol 1997; 24: 115768.
Franco AA, Cheng R, Chung G-T, Wu S, Sears CL. Molecular
evolution of the pathogenicity island of enterotoxigenic
Bacteroides fragilis strains. J Bacteriol 1999; 18: 6623-33.
van Putten AJ, Jochems GJ, de Lang R, Nijkamp HJJ. Structure
and nucleotide sequence of the region encoding the mobilization
proteins of plasmid CloDF13. Gene 1987; 51: 171-8.
Giacomini A, Squartini A, Nuti MP. Nucleotide sequence and
analysis of plasmid pMD136 from Pediococcus pentosaceus
FBB61 (ATCC43200) involved in Pediocin A production. Plasmid
2000; 43: 111-22.
Tauch A, Schneiker S, Selbitschka W, Puhler A, Van Overbeek LS,
Smalla K, et al. The complete nucleotide sequence and
environmental distribution of the cryptic, conjugative, broad-host-
Current Pharmaceutical Design, 2004, Vol. 10, No. 13 1565
[202]
[203]
[204]
[205]
[206]
[207]
[208]
[209]
[210]
[211]
[212]
range plasmid pIPO2 isolated from bacteria of the wheat
rhizosphere. Microbiology 2002; 148: 1637-53.
Godon JJ, Pillidge CJ, Jury K, Gasson MJ. Characterization of an
original conjugative element - the sex factor of Lactococcus lactis
712. Lait 1996; 76: 41-9.
Takai S, Hines SA, Sekizaki T, Nicholson VM, Alperin DA, Osaki
M, et al. DNA sequence and comparison of virulence plasmids
from Rhodococcus equi ATCC 33701 and 103. Infect Immun 2000;
68: 6840-7.
Okinaka R, Cloud K, Hampton O, Hoffmaster A, Hill K, Keim P,
et al. Sequence, assembly and analysis of pX01 and pX02. J Appl
Microbiol 1999; 87: 261-2.
Stedman KM, She Q, Phan H, Holz I, Singh H, Prangishvili D,
et al. pING family of conjugative plasmids from the extremely
thermophilic archaeon Sulfolobus islandicus: insights into
recombination and conjugation in crenarchaeota. J Bacteriol 2000;
182: 7014-20.
She Q, Phan H, Garrett RA, Albers SV, Stedman KM, Zillig W.
Genetic profile of pNOB8 from Sulfolobus: the first conjugative
plasmid from an archaeon. Extremophiles 1998; 2: 417-25.
Cloeckaert A, Baucheron S, Chaslus-Dancla E. Nonenzymatic
chloramphenicol resistance mediated by IncC plasmid R55 is
encoded by a floR gene variant. Antimicrob Agents Chemother
2001; 45: 2381-2.
Begg KJ, Dewar SJ, Donachie WD. A new Escherichia coli cell
division gene, ftsK. J Bacteriol 1995; 177: 6211-22.
Krall L, Wiedemann U, Unsin G, Weiss S, Domke N, Baron C.
Detergent extraction identifies different VirB protein subassemblies
of the type IV secretion machinery in the membranes of
Agrobacterium tumefaciens. Proc Natl Acad Sci USA 2002; 99:
11405-10.
Frankel AE, Koo HM, Leppla SH, Duesbery NS. and Wounde GF.
Novel protein targeted therapy of metastatic melanoma. Curr
Pharm Des 2003; 9(25): 2060-6.
Ibrahim HR, Aoki T. and Pellegrini A. Strategies for new
antimicrobial proteins and peptides: lysozyme and aprotinin as
model molecules. Curr Pharm Des 2002; 8(9): 671-93.
Ilies MA, Seitz WA. and Balaban AT. Cationic lipids in gene
delivery: principles, vector design and therapeutical applications.
Curr Pharm Des 2002; 8(27): 2441-73.