Download F factor conjugation is a true type IV secretion system

FEMS Microbiology Letters 224 (2003) 1^15
www.fems-microbiology.org
MiniReview
F factor conjugation is a true type IV secretion system
T.D. Lawley, W.A. Klimke, M.J. Gubbins, L.S. Frost
Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada T6G 2E9
Received 18 March 2003; received in revised form 15 May 2003; accepted 16 May 2003
First published online 14 June 2003
Abstract
The F sex factor of Escherichia coli is a paradigm for bacterial conjugation and its transfer (tra) region represents a subset of the type
IV secretion system (T4SS) family. The F tra region encodes eight of the 10 highly conserved (core) gene products of T4SS including
TraAF (pilin), the TraBF , -KF (secretin-like), -VF (lipoprotein) and TraCF (NTPase), -EF , -LF and TraGF (N-terminal region) which
correspond to TrbCP , -IP , -GP , -HP , -EP , -JP , DP and TrbLP , respectively, of the P-type T4SS exemplified by the IncP plasmid RP4.
F lacks homologs of TrbBP (NTPase) and TrbFP but contains a cluster of genes encoding proteins essential for F conjugation (TraFF ,
-HF , -UF , -WF , the C-terminal region of TraGF , and TrbCF ) that are hallmarks of F-like T4SS. These extra genes have been implicated in
phenotypes that are characteristic of F-like systems including pilus retraction and mating pair stabilization. F-like T4SS systems have been
found on many conjugative plasmids and in genetic islands on bacterial chromosomes. Although few systems have been studied in detail,
F-like T4SS appear to be involved in the transfer of DNA only whereas P- and I-type systems appear to transport protein or
nucleoprotein complexes. This review examines the similarities and differences among the T4SS, especially F- and P-like systems, and
summarizes the properties of the F transfer region gene products.
= 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Conjugation; Plasmid; Type IV secretion ; Pili; Membrane complex
1. Introduction
In 1946, Joshua Lederberg proposed that a ‘‘cell fusion
would be required’’ to facilitate the transfer of F factor
DNA, integrated in the chromosome of the donor cell,
into recipient Escherichia coli [1]. We now know that
this cell fusion is constructed by the type IV secretion
system (T4SS) encoded on Gram-negative conjugative elements. T4SS, also known as the mating pair formation
(Mpf) apparatus, are central to the dissemination of numerous genetic determinants between bacteria, as highlighted by the spread of antibiotic resistance among pathogens [2,3]. T4SS are cell envelope-spanning complexes (11^
13 core proteins) that are believed to form a pore or
channel through which DNA and/or protein travels from
the cytoplasm of the donor cell to the cytoplasm of the
recipient cell. T4SS have also been found to secrete virulence factor proteins directly into host cells as well as take
* Corresponding author. Tel. : +1 (780) 492-0672;
Fax : +1 (780) 492-9234.
E-mail address : [email protected] (L.S. Frost).
up DNA from the medium during natural transformation,
revealing the versatility of this macromolecular secretion
apparatus [4,5]. Despite the clinical and evolutionary importance of T4SS, the general mechanism by which they
secrete or take up macromolecules remains unknown.
The F factor remains a paradigm for understanding the
mechanism by which T4SS transfer macromolecules across
the membranes of Gram-negative bacteria [6^8]. DNA
transfer occurs within the tightly appressed cell envelopes
of mating cells, which are referred to as conjugation junctions [9^11]. These junctions form in the presence of the
Mpf or T4SS proteins; the same proteins that assemble
pili (Figs. 1 and 2 ; Table 1) and transfer DNA. Conjugation is thought to be initiated by contact between the
F-pilus and a suitable recipient resulting in pilus retraction
[12] and stable mating pair or aggregate formation [9].
Prior to the initiation of DNA transfer, the relaxosome,
consisting of proteins bound to the origin of transfer
(oriT), resides within the cytoplasm of donor cells [13].
A mating signal, possibly generated by contact between
the pilus and recipient cell, appears to result in a speci¢c
interaction between the relaxosome and the coupling protein, or nucleic acid pump, at the inner face of the con-
0378-1097 / 03 / $22.00 = 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00430-0
FEMSLE 11051 27-6-03
2
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
Fig. 1. Comparison of F-like T4SS with each other and with P- and I-like T4SS. Transfer genes are presented with color/pattern, with the same color/
pattern representing homologous gene products (see Table 1), while non-essential transfer genes are white. Light gray genes represent transfer gene
products with no shared homology to other T4SS subfamilies. Lipo = lipoprotein motif ; band within arrow = Walker A motif; upper case gene names = Tra; lower case gene names = Trb (F, pNL1 and RP4) or Trh (R27). Double slash indicates non-contiguous regions. The gene sizes are relative to
each other. Maps were produced using the indicated GenBank accession numbers: F-NC_002483; pED208-AY046069 ; R27-NC_002305; Rts1NC_003905; pNL1-NC_002033; R391-AY090559; SXT-AY055428; RP4-NC_001621; R64-AB027308. See text for details and references.
jugative pore [14,15]. Coupling protein^relaxosome contact could lead to DNA unwinding, generating a single
strand of DNA that is then transferred to the recipient
in a 5P to 3P direction [16^18]. This two-step mechanism
has been proposed to result in the transport of the relaxase, covalently bound to the 5P end of the transferring
strand (T-strand), into the recipient through the T4SS
conjugative pore [19]. The detection of the relaxase in
the recipient has as yet not been successful, however, the
topological constraints of DNA transfer combined with
the role of the relaxase in termination makes this highly
probable. Considerable circumstantial evidence supports
the transfer of a pilot protein, such as the relaxase, along
with the DNA. The most compelling is the indirect evidence for transport of a primase, encoded as a domain of
the relaxase protein by the IncQ mobilizable plasmid
R1162, that could initiate replacement DNA strand synthesis in the new transconjugant [20]. Interestingly, the
IncP and I conjugative systems also transport primase
molecules either alone or in conjunction with the DNA
[21,22], suggesting an evolutionary relationship with the
IncQ system. The transport of the VirD2^T-DNA com-
FEMSLE 11051 27-6-03
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
3
Fig. 2. A representation of the F-like T4SS transfer apparatus drawn from available information. The pilus is shown assembled with ¢ve TraA (pilin)
subunits per turn extending from the inner membrane through a putative secretin-like outer membrane pore involving TraK. TraK is anchored by
TraV and interacts with TraB in the inner membrane. TraB interacts with the coupling protein creating a continuous pore from the cytoplasm through
the cell envelope to the extracellular environment. Other components of the inner membrane and periplasm are indicated, with TraL and TraE seeding
the site of pilus assembly and attracting TraC to the pilus base where it acts to drive assembly in an energy-dependent manner. The Mpf proteins include TraG and TraN that aid in mating pair stabilization (Mps). TraF, -H, -U, -W and TrbC, together with TraN, are speci¢c to F-like systems and
might have a role in pilus retraction, pore formation and mating pair stabilization.
plex from Agrobacterium tumefaciens to wounded plant
tissue to initiate crown gall formation is another example
of a relaxase-like protein bound to the 5P end of a singlestranded DNA molecule being transported via a T4SS
[23^25]. Thus it is not impossible to think of conjugative
DNA transfer as a protein transport system that has been
modi¢ed to transfer DNA along with a protein substrate.
The core T4SS proteins in F, TraAF (pilin), -LF , -EF ,
-KF , -BF , -VF , -CF and -GF (N-terminal domain), also
require the auxiliary, essential gene products TraFF , -GF
(C-terminal domain), -HF , -NF , -UF , -WF and TrbCF for
pilus assembly and mating pair stabilization. Additional
essential gene products in the F conjugative system include
the coupling protein, TraDF , and the members of the relaxosome, TraIF , a relaxase^helicase bifunctional protein,
TraMF , and TraYF that are required for DNA transfer.
TraBF along with TraCF are the quintessential T4SS
proteins and are the easiest to ¢nd homologs for in
BLAST searches. Similarly, the coupling protein (e.g.
TraDF ) is the signature homolog of conjugative T4SS systems capable of nucleic acid transport [19], whereas
TrbBP /VirB11Ti /TraJI homologs are indicative of P-type/
Ti/I-type systems [26]. The auxiliary genes present in F
(encoding TraFF , -GF (C-terminal domain), -HF , -NF ,
-UF , -WF and TrbCF ) are conserved throughout F-type
systems and serve as hallmarks of this family. These
gene products are essential for F transfer and appear to
be involved in pilus retraction and mating pair stabilization, which are critical factors for e⁄cient F conjugation
in liquid media. The conjugative ability of P-type systems,
which lack these homologs, is lower in liquid media than
on solid media and may re£ect the di¡erent ecological
niches inhabited by bacteria carrying the F- and P-type
transfer systems [27].
The proteins involved in conjugal DNA metabolism as
well as those involved in the regulation of gene expression
or the prevention of conjugation between donor cells (surface and entry exclusion, TraTF and -SF , respectively) will
not be discussed here. The interested reader is directed to
reviews by Lanka and Wilkins [28], Lawley et al. [8], Llosa
et al. [19] and Zechner et al. [18]. This review will discuss
the essential T4SS proteins in F-type systems (IncF, IncHI, IncJ, IncT and the SXT element, among others),
which di¡er in signi¢cant ways from P-type systems such
as that of RP4 (IncP), Ptl (Bordetella pertussis toxin excretion system) and VirB (Ti plasmid tumorigenesis system
of A. tumefaciens) T4SS [2,4] (see below). A third system,
the I-type, about which relatively little is known, is exempli¢ed by the IncI plasmid T4SS that have signi¢cant homology to the virulence factor transport systems of Legionella pneumophila [29^31].
2. F-like T4SS components
The essential components of the F-like T4SS are de¢ned
as those Mpf proteins that are essential for conjugation, as
determined by mutagenesis and complementation experiments of both the F factor and the IncHI1 plasmid R27
[7,32^34]. Results obtained from investigations into individual Mpf proteins from both the F factor and the R27
T4SS are combined to create an F transfer protein family
FEMSLE 11051 27-6-03
4
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
Table 1
Summary of conserved F-like T4SS components
Proteina P-type
homologd
I-type
homologd
Size rangee Signal
(aa)
sequencef
Cellular
Motifsh
g
location(s)
TraX
112^128
93^105
130^261
299^410
429^475
171^316
799^893
912^1329
Y
N
Y
Y
N
Y
N
N
IM, E
IM
IM/P
P/OM
IM/P
OM
IM
IM/P
150^368
210^502
203^254
330^358
602^1230
N
Y
Y
Y
Y
IM
P
P
P
OM
TraF
257^363
Y
P
TraH
Orf169
TrbB
453^501
169^265
230^298
Y
Y
Y
OM
IM
P
TraA
TraL
TraE
TraK
TraB
TraV
TraC
TraG
TrbC/VirB2
TrbD/VirB3
TrbJ/VirB5
TrbG/VirB9
TrbI/VirB10
TrbH/VirB7
TrbE/VirB4
TrbL/VirB6
TrhPb
TraWc
TrbC
TraU
TraN
TraF
TraN
TraO
TraI
TraU
TrbB
Proposed function
Pilin
Pore
Pore
Secretin
Pore
Coiled-coil Pore
Lipoprotein Pore
ATPase
Secretion
Mating pair
stabilization ; pore
Peptidase
Transfer peptidase
Pore
Pore
DNA transfer
Cysteine-rich Mating pair
stabilization ; adhesin
Disul¢de
Disul¢de bond
isomerase
formation
Coiled-coil Pore
Lysozyme
Transglycosylase
Disul¢de
Disul¢de bond
isomerase
formation
Interacting partners in
F- and P-like T4SSi
Interaction
reference
TraXF , TraQF
TrhCH
TrhCH
TraBF , TraVF
TraKF , TrhCH ,, TraGH
TraKF
TrhBH , TrhEH , TrhLH
TraSF
[41]
[34]
[34]
[40]
[34,40]
[40]
[34]
[2]
TrbC
TraW
Components in bold indicate homology to P-like T4SS.
a
Nomenclature according to the F system except for the peptidase TrhP which is named according to R27 nomenclature. The R27 transfer protein nomenclature is Trh [33,34]. The P- and I-type nomenclature is according to Christie [2].
b
R27, Rts1, R391, SXT and pNL1 systems contain a peptidase with the peptidase of pNL1 containing an N-terminal fusion to TrbI, suggesting a
coupled function. F and pED208 do not contain peptidases.
c
In R27, Rts1, R391 and SXT systems TrbC is fused to the N-terminus of TraW, suggesting a coupled function, whereas they are separate proteins in
F and pED208.
d
Homology deduced based on similarity identi¢ed with PSI-BLAST analysis or functional analogy.
e
Range is determined by comparing homologs in F, pED208, R27, Rts1, R391, SXT and pNL1.
f
Signal sequence predicted with SignalP (http://www.cbs.dtu.dk/services/SignalP).
g
Inner membrane (IM), periplasm (P), outer membrane (OM) and extracellular (E).
h
Motifs identi¢ed with ScanProsite (http://ca.expasy.org/tools/scanprosite/), CDD (http://www.ncbi.nlm.nih.gov/BLAST/) or Coils (http://www.ch.
embnet.org/software/COILS_form.html).
i
Transfer system in which direct or indirect interaction identi¢ed is indicated with subscript.
in this overview, as it is likely that homologs are functionally equivalent. Based on work on the F factor, the T4SS
proteins are organized according to three proposed functions : (1) pilin and pilin processing, (2) pilus tip formation
and pilus extension and (3) mating pair stabilization [7]
(Figs. 1 and 2; Table 1). Other non-essential components
of F-like T4SS are TraP, a protein that stabilizes the extended pilus; TrbB, a putative thioredoxin homolog ; TrbI,
a protein which promotes DNA transport and has homology to the FliK £agellum assembly protein (L.S. Frost,
unpublished results); and Orf169, a lytic transglycosylase
with homologs in P- and I-like systems.
2.1. F-like propilin processing
The propilin subunits from F-like T4SS ranges in size
from 112 to 128 aa (Table 1). The pilin subunit is poorly
conserved among T4SS, for example the pilin subunit of
the R27 (TrhAH ) shares more similarity with the IncP
pilin (TrbCP ) than with the IncF pilin (TraAF ) [34]. All
F-like propilin subunits contain a long leader sequence
that is either known or predicted to be cleaved by the
host leader peptidase, LepB, to produce a peptide of 68^
78 aa [35,36]. After removal of the signal sequence, the
F-pilin subunit is oriented in the inner membrane with
its N- and C-termini positioned in the periplasm [37,38].
Indeed, all pilin subunits of the F-type T4SS appear to
contain two hydrophobic regions that serve as transmembrane regions. The correct insertion and accumulation of
F-pilin in the inner membrane requires the chaperone-like
inner membrane protein, TraQ, which is present only in
T4SS closely related to F itself [39]. Pilin subunits typically
undergo an additional processing reaction, which has been
identi¢ed as acetylation by TraXF in F-like pilins (F, R1,
R100-1, pED208) [39^41] or cyclization by the peptidase
TraFP in P-like pilins (RP4 and Ti) [42]. The propilin subunits of R27, Rts1, R391, SXT and pNL1, which are
encoded by F-like T4SS, are more similar to P-like pilins.
They are likely cleaved at the C-terminus and possibly
cyclized by a transfer peptidase/cyclase, although this has
yet to be demonstrated.
Pilin insertion into the membrane and maturation are
FEMSLE 11051 27-6-03
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
the ¢rst steps in pilus production. Assembly of conjugative
F-like pili on the bacterial surface requires the remainder
of the T4SS and the auxiliary gene products, except for the
C-terminal domain of TraG, TraN and TraU. F-pilin subunits are stored as a pool in the inner membrane prior to
assembly on the cell surface [43]. Pili are assembled by
addition of pilin subunits to the base of the pilus, as demonstrated by H-pili of R27 [44]. In response to contact
with a suitable recipient, pilus retraction appears to proceed in an energy-independent manner [40], which is the
reverse of assembly, whereby the pilin subunits return to
the membrane and possibly serve to stabilize the mating
pair or be a part of the conjugative pore.
Homology studies have revealed that the pilin gene appears to have been shu¥ed among various T4SS during
evolution. For example, the IncHI1 plasmid, R27, has an
F-like T4SS except for a P-like pilin protein and corresponding peptidase/cyclase [34]. The lack of sequence conservation in pilin could be due to: (1) rapid evolution of
the pilin subunits in response to strong selective forces of
extracellular factors such as phage and receptors on recipients and (2) lateral gene transfer of F-, P- and I-like
propilin and processing genes between T4SS subfamilies.
In fact, the cassette-like nature for the development of the
T4SS is striking and suggests that there has been considerable opportunity for ‘mix and match’ during evolution.
2.2. F-like T4SS pilus assembly
Mutations in traL, -E, -K, -B, -V, -C, -W, -F, -H, and
the 5P end of traG have broadly similar phenotypes, which
include the inability to assemble pili and transfer DNA [7].
Using a sensitive M13K07 transducing phage assay, Anthony et al. [32] identi¢ed two mutant subgroups that are
consistent with two steps in pilus assembly : (a) those mutations that prevent pilus tip formation on the cell surface
(in traL, -E, -K, -C, -G) and (b) those that allow tip formation but block pilus extension (traB, -V, -W, -F, -H).
These results provided the ¢rst example in any T4SS of a
di¡erentiation of roles for Mpf proteins. The F-like T4SS
components will be organized according to these results.
2.2.1. Pilus tip formation
2.2.1.1. TraLF . Members of the TraLF family range in
size from 93 to 105 aa and are homologous to TrbDP (103
aa) and VirB3Ti (108 aa) [45]. TraL is predicted to localize
to the inner membrane, as is TrbDP [46]. In F, TraLF has
never been visualized, suggesting it could be the limiting
factor determining the number of F-pili per cell. TrhLH ,
along with TrhEH and TrhBH , of R27 (IncHI1) was shown
to be essential for the formation of TrhCH complexes,
indicating either a direct or an indirect interaction between
TrhLH and TrhCH [47].
2.2.1.2. TraEF .
TraEF family members range in size
5
from 130 to 261 aa and are homologous to TrbJP (258
aa) and VirB5Ti (220 aa) [7]. TraEF and TrbJP are predicted to be located in the inner membrane (RP4) [7,46]
whereas VirB5Ti is thought to be a minor component of
the T-pilus [48].
2.2.1.3. TraKF . The TraKF family of proteins range in
size from 299 to 410 aa and are homologous to TrbGP
(297 aa), VirB9Ti (293 aa) and TraNI (327 aa) [7]. TraKlike proteins are predicted to be located in the periplasm
or outer membrane [7,46,49]. This protein family shares
similarity to secretin proteins, especially the HrcC subgroup of the type III secretion system (T3SS) encoded
by Pseudomonas syringae [50] (Fig. 3). The C-terminal
regions of TraKF proteins are conserved in both the
L-domain and S-domain of the prototypical secretin
PulD of Klebsiella oxytoca [34]. The L-domain is present
in all secretins and is proposed to be embedded within the
outer membrane to form the ring structure typical of secretins. The S-domain is a region of 60 aa that binds to a
lipoprotein which serves as a periplasmic chaperone [51].
The C-terminus of TraKF has been shown to interact with
TraVF , a lipoprotein, and the N-terminus of TraKF interacts with TraBF , an inner membrane protein [49]. The
TraBF -TraKF -TraVF complex likely forms an envelopespanning structure similar to that of VirB10-VirB9-VirB7
of the Ti plasmid T4SS [52]. Although TraKF is a periplasmic protein, it associates with the outer membrane in
the presence of the F T4SS [49]. The presence of a putative
secretin within the T4SS suggests a mechanism by which
both the pilus and DNA could transverse the outer membrane.
2.2.1.4. TraCF . Members of the TraCF family of proteins range in size from 799 to 893 aa and are homologous
to TrbEP (852 aa), VirB4Ti (788 aa) and TraUI (1014 aa).
TraCF is predicted to be a peripheral inner membrane
protein whose localization is dependent upon the presence
of the T4SS, speci¢cally TraLF [47,53]. All members of
this protein family contain both Walker A and Walker
B motifs, which energize pilus assembly [54,55]. A point
mutation in traCF , traC1044, is a temperature-sensitive
mutation that blocks pilus assembly [56]. Using TrhCGFP fusions, TrhCH of R27 was shown to form complexes in the inner membrane, possibly containing other
transfer proteins. The formation of TrhC-GFP complexes
was dependent on the presence of TrhBH , -EH and -LH ,
suggesting either a direct or an indirect interaction between these proteins [47].
2.2.1.5. TraGF . TraGF proteins range in size from 913
to 1329 aa. TraGF proteins have two roles in conjugation:
the N-terminal region is involved in pilus tip formation
and pilus assembly whereas the entire protein is involved
in mating pair stabilization [57] (see below). The N-terminal 500^600 aa is proposed to be localized to the inner
FEMSLE 11051 27-6-03
6
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
VcoSXTTraK
PreR391TraK
2.2.2. Pilus extension
100
SmeHtdP
PvuRts1TraK
StyR27TrhK
100
84
89
NarpNL1TraK
StypED208TraK
70
100
100
100
100
PsysyringaeHrpH
58
100
PsytomatoHrcC
StyR100TraK
EcoFTraK
EcoColB2TraK
StypSLTTraK
100
86
99
PcaHrcC
PchHrcC
PflRscC
PstHrcC
EamHrcC
500 changes
Fig. 3. Phylogenetic analysis of T4SS secretin-like proteins of the TraKF
family with the HrcC T3SS secretin family. A PSI-BLAST search of the
non-redundant bacterial database was used to generate the alignment.
The expect size was 100^1000 with gap open and gap extension penalties of 7 and 2, respectively. The sequences were multiply aligned using
ClustalX with a Gonnet matrix; gap open and gap extension penalties
were set within the range 4^10 and 0.2^0.5, respectively. Sequences with
very little divergence were discarded and the multiple alignment was imported into MacClade v4.0 to generate a *.nexus ¢le. Phylogenetic analysis was carried out with the PAUP 4.0 beta 8/10 software package using parsimony and a heuristic search of 100 replicates with the PAM250
(modi¢ed matrix) character type. Once a ¢nal tree was selected, it was
bootstrapped through 100 replicates to give the consensus values shown.
The various homologs are listed on the tree using a three-letter abbreviation for the bacterial host, followed by the plasmid name (if applicable) and the gene or protein name. The following list comprises all of
the relevant information in the homology tree in the following format:
three-letter bacterial species abbreviation, plasmid name (if applicable),
gene or protein name, accession number, full bacterial species name. Pre
R391 TraK AAM08021 P. rettgeri; Vco SXT TraK AAL59718 V. cholerae ; Pvu Rts1 orf209 BAB93771 P. vulgaris; Nar pNL1 TraK
NP_049166 N. aromaticivorans; Psy_syr HrpH AAC05014 P. syringae
pv. syringae; Psy_tom HrcC AAC34756 P. syringae pv. tomato ; Pca
HrcC AAK97280 Pectobacterium carotovorum subsp. carotovorum ; Pch
HrcC AAC31975 Pectobacterium chrysanthemi ; Eam HrcC AAB49179
Erwinia amylovora; Pst HrcC AAG01463 Pantoea stewartii subsp. stewartii ; P£ RscC AAK81929 Pseudomonas £uorescens; Sty pSLT TraK
NP_490566 S. typhimurium; Eco ColB2 TraK AAB07774 E. coli; Eco F
TraK AAC44189 E. coli ; Sty R100-1 TraK AAB07769 S. typhi; Sty
pED208 TraK AAM90706 S. typhi ; Sty R27 TrhK AAD54050 S. typhimurium ; Sme R478 HtdP AAL27020 Serratia marcescens.
membrane and contains six to eight transmembrane regions whereas the remaining C-terminal region is located
within the periplasmic space (unpublished results). The
N-terminal domain is homologous with TrbLP (528 aa)
and VirB6Ti (295 aa), both of which are also predicted
to contain multiple transmembrane regions and are essential for pilus biosynthesis (Fig. 4).
2.2.2.1. TraBF . Members of the TraBF protein family
range in size from 429 to 475 aa and are homologous to
TrbIP (463 aa), VirB10Ti (377 aa) and TraOI (429 aa)
within their C-terminal regions (Fig. 5). Limited homology
also exists among all these homologs with FliF, a structural protein in the type III secretion system involved in
£agellum assembly (L.S. Frost, unpublished observations)
[58]. TraBF -like proteins are predicted to contain an
N-terminal anchor with the bulk of the protein located
within the periplasm. The N-terminal region of F-like
TraB proteins contain coiled-coil domains, which are
probably involved in multimerization, and a proline-rich
domain, suggesting an extended structure [59]. The proline-rich domain, by analogy with other such motifs, could
interact with SH3 domains in other proteins, an interaction central to signal transduction [60]. TrhBH of R27 was
recently shown to interact with itself and with the coupling
protein TraGH [59] providing exciting evidence that the
T4SS and the coupling protein (in F, TraDF ) do, indeed,
‘couple’, linking the relaxosome to the T4SS.
2.2.2.2. TraFF . The TraFF protein family ranges in
size from 257 to 363 aa and shares homology to TrbBF
(a non-essential, conserved, F-like T4SS component) and
TrbBI of the IncI transfer system; there is no known homolog in P-like systems. These proteins share similarity to
the thioredoxin superfamily, characterized by the C-X-XC motif and the thioredoxin fold (Elton et al., in preparation). Since TraF is localized to the periplasmic space,
these proteins likely play a role in thiol redox chemistry
within the periplasm, possibly involving disul¢de bond
formation or isomerization. It is interesting to note that
several T4SS components localizing to the periplasm contain multiple, conserved cysteine residues including homologs of TraBF , -PF and -GF (2), TraHF and -VF (3),
TraUF (10) and TraNF (22) with homologs of TrbIF having a single conserved cysteine [8,61]. In addition, members of this protein superfamily have also been proposed
to act as chaperones that prevent inappropriate interactions with other proteins [62]. In light of these observations, perhaps TraFF - and TrbBF -like proteins are key to
the disul¢de bond chemistry in F-like T4SS assembly.
2.2.2.3. TraHF . TraHF -like proteins range in size
from 453 to 501 aa and are unique to the F-like T4SS
subfamily. Members of the TraHF protein family are localized to the periplasm/outer membrane [63] and contain
C-terminal coiled-coil domains, suggesting the formation
of higher order structures, either with other TraHF molecules or with other components of the T4SS.
2.2.2.4. TraWF -TrbCF . Members of the TraWF protein family range in size from 210 to 502 aa and are unique
to the F-like T4SS subfamily. TrbC is fused to the N-ter-
FEMSLE 11051 27-6-03
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
minus of TraW in R27, Rts1, R391 and SXT, whereas
TraW and TrbC are separate proteins in F, pED208 and
pNL1. The fusion of TrbCF to TraWF suggests that the
functions of these proteins are linked. Both proteins are
proposed to be localized to the periplasmic space. TrbCF
is correctly processed in the presence of TraNF suggesting
a relationship between these two proteins, which are encoded on adjacent genes in the F tra operon [64].
2.2.2.5. TraVF . TraVF -like proteins range in size from
171 to 316 aa and are lipoproteins with a signature cysteine at the processing site [65]. Although TraVF proteins
share little similarity to TrbHP (160 aa), VirB7Ti (55 aa) or
TraII (272 aa) beyond two conserved cysteines thought to
be involved in multimerization, they appear to be functional analogs that interact with secretin-like proteins
such as TraKF and VirB9Ti . Indeed, TraVF has been
shown to interact with TraKF , a putative secretin [49],
and VirB7Ti is known to interact with VirB9Ti [66,67]
2.2.3. Mating pair stabilization
Mating pair stabilization is a unique feature of F-like
T4SS and is believed to be at least partially responsible for
facilitating DNA transfer in liquid environments. Based
on the experimental evidence of Kingsman and Willetts
[68] and recent evidence involving TraGF in recognition
of the TraSF entry exclusion protein (L.S. Frost, unpublished results), mating pair stabilization might involve
building a structure between the two cells that ‘staples’
them together. Mating pairs are di⁄cult to break apart
prematurely and require signi¢cant force to do so. However, about 30 min after the start of F plasmid transfer,
the cells spontaneously separate suggesting an active
mechanism involving the expression of genes in the new
transconjugant, previously identi¢ed as being in the distal
part of the F tra operon [69]. Candidates for mating pair
separation include the entry and surface exclusion proteins
TraSF and TraTF as well as the relaxase (TraIF ) and coupling protein (TraDF ), which might generate a break in
DNA transport signalling the termination of conjugation.
2.2.3.1. TraGF . The whole of TraGF , but especially
the C-terminal region, is involved in mating pair stabilization. In F T4SS, the C-terminal region is fused to a homolog of TrbLP /VirB6Ti suggesting that these homologs
might be involved in forming a conjugative pore with
varying degrees of sturdiness. This region is predicted to
be located within the periplasmic space and has been proposed to interact with TraNF to stabilize mating pairs [57].
A second C-terminal product TraG*, which is believed to
be a cleavage product of the full-length protein, has been
detected in the periplasm [57] although its importance in
transfer is in doubt (L.S. Frost, unpublished results).
TraGF is involved in entry exclusion, a process by which
DNA synthesis and transport from the donor cell is
blocked by TraSF in the inner membrane of the recipient
7
cell. TraGF could be translocated to the recipient cell
where it would interact with TraSF instead of its true
receptor. TraGF is plasmid-speci¢c for TraSF and this
speci¢city maps to a central C-terminal domain of TraGF
(L.S. Frost, unpublished observation). If homologs of
TraGF are involved in mating pair junction formation, it
suggests that the periplasmic space of the donor cell contracts bringing the inner and outer membrane together. In
P-type systems, the TrbLP /VirB6Ti homologs might not be
able to penetrate the cell envelope of the recipient cell, a
function of the pilus, whereas the C-terminal domain of
TraGF homologs reaches all the way to the inner membrane of the recipient to stabilize the pilus penetration
event.
2.2.3.2. TraNF . TraNF -like proteins are 602^1230 aa
and are unique to F-like T4SS; they are signature proteins
for the auxiliary class of T4SS that de¢ne the F-like subfamily [70]. This family of proteins appear to act as ‘adhesins’ based on evidence for TraNF which is present in
the outer membrane of donor cells. TraNF of the F plasmid interacts with the major outer membrane protein
OmpA in recipient cells to stabilize the mating pairs prior
to DNA transfer. Other F-like TraNF proteins do not
necessarily interact with OmpA, for instance, TraNR100
of the F-like R100 plasmid does not share this receptor.
The N- and C-terminal regions of TraNF proteins are
highly conserved whereas the central region displays extensive divergence. It is this central region that is involved
in OmpA recognition by TraNF as well as TraNF multimerization [71]. Preliminary evidence suggests that TraNF
and TraVF interact since some mutants of traN are destabilized in the absence of traV [70].
2.2.3.3. TraUF . Members of the TraUF protein family
range in size from 330 to 358 aa and are unique to the
F-like T4SS subfamily. TraUF is a periplasmic protein
that is essential for DNA transfer but not formation of
conjugative pili, as 20% of donors containing F traU mutations produce pili. TraUF is therefore proposed to be
primarily involved in DNA transfer perhaps by aiding
mating pair stabilization and conjugative pore formation
since mutations in traU, -G and -N have the same phenotype [72].
3. Relationships between F- and P-type T4SS
It has been long been recognized that there are two
types of conjugative pili: long, £exible pili and short, rigid
pili [27]. It is now evident that long, £exible pili are encoded by F-type T4SS (IncF, -H, -T, -J) whereas short,
rigid pili are encoded by P-type T4SS (IncP, -N, -W, -I).
The long, £exible pili produced by F-like T4SS measure 2^
20 Wm and have a diameter of 8 nm with a central lumen
measuring 2 nm. The pilin subunits are arranged as a he-
FEMSLE 11051 27-6-03
8
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
lical array. F-pili are easily seen attached to cells and
appear £exible in electron micographs. The short, rigid
pili produced by P-like T4SS are seldom seen attached
to donors. They measure 8^12 nm in diameter [42] and
are usually under 1 Wm in length. No information on the
arrangement of the circular subunits in the assembled pilus
is currently available for P-like pili. The di¡erences in pilus
structure are not likely dictated by di¡erences in pilin processing, such as acetylation or cyclization, since acetylase
and transfer peptidase coding regions can be present in
Atu pAT VirB6
Atu pAT AvhB6
Ret VirB6
Sme pSymA VirB6
Lpn LvhB6
Lpn LvhB6
Xax pXA64 VirB6
Ppu pWWo MpfE
pIPO2T TraH
Sme pSB102 TraH
Xca VirB6
Xca VirB6
Xca VirB6
Xca VirB6
Xax VirB6
Xax VirB6
Rrh pRi1724 riorf158
Atu pTiAB2 VirB6
Atu pTi VirB6
Atu pTiC58 VirB6
Atu pRiA VirB6
Atu pTi VirB6
Hpy jhp0033
Hpy orf1
Rrh pRi1724 riorf124
Atu pRiA4b TrbL
Rrh pNGR234a TrbL
Atu pTi AGR_pTi_76p
Atu pTiSakura tiorf11
Atu pTi TrbL
Psp pADP1 TrbL
Cte pTSA TrbL
Eae R751 TrbL
Psp pB4 TrbL
RK2 TrbL
Xfa pXF51 Xfa0037
Mlo mlr6402
Mlo TrbL
Mlo pMLb m119606
Rso TrbL
Sen pHCM1 HCM1.262
Sty R27 TrhG
Sfl R100 TraG
Eco F TraG
Sty pSLT TraU
Sty pED208 TraG
Vco SXT TraG
Pre R391 TraG
Rco RC0146
Rpr RP108
Eco R388 TrwI
Sen SO11
Eco R6K PilX6
Ype pYC orf5
Sty R46 TraD
Xfa pXF51 Xfa0011
Mlo pMLa mlr9255
Mlo R71 msi411
Ngo_JC1 TraG
Pvu Rts1 orf242
Bhe VirB6
Cac CAC2046
Nar pNL1 TraG
Eco R721 TraA
Ccr CC2420
Bpe VirB6
Hpy jhp0937
Sco SCD72A.15c
Ban pX01 pX01-79
Bf1 hydrophobic protein
Cac CAC2403
Bth TraJ
Nsp alr7534
FEMSLE 11051 27-6-03
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
both F-like and P-like T4SS. Instead, the di¡erences probably lie with the auxiliary genes in F-like systems or TrbBP
in P-like systems, which de¢ne these two groups (see below).
Long, £exible pili allow donors to mate in liquid and on
solid media with approximately equal e⁄ciencies whereas
short, rigid pili result in a surface-preferred mating phenotype [27,73]. Long, £exible pili likely retract and allow
mating pair stabilization thereby facilitating mating in liquid media, a property not available to systems with short,
rigid pili. Retraction [12,74,75] is reminiscent of type IV
pili encoded by type II secretion systems [76,77] and is
proposed to occur in response to a ‘mating signal’ received
from the pilus tip when it contacts a suitable recipient cell.
Other conjugative elements which contain F-type T4SS
include, besides the F factor (E. coli) [7], R100 (IncFII)
[32], pED208 (IncFV; S. typhi) [39], R27 (IncHI1; S. typhi) [78], Rts1 (IncT; Proteus vulgaris) [79], R391 (IncJ;
Providencia rettgeri) [80], SXT element (Vibrio cholerae)[81] and pNL1 (Novosphingomonas aromaticivorans)
[82] (Fig. 1). Neisseria gonorrhoeae contains an F-type
T4SS that is not used for conjugation, but rather for the
secretion of DNA [83]. It is interesting that no F-type
T4SS have been reported to secrete virulence factors. In
fact, no F-type T4SS to date has been shown to secrete
proteins [21].
Conjugative elements that contain P-type T4SS include
RP4 (IncPK; Pseudomonas aeruginosa) [84], R751 (IncPL;
Klebsiella aerogenes) [85], pKM101 (IncN; Salmonella
typhimurium)[86] and R388 (IncW; E. coli; accession num-
9
ber X81123) (see [4]). In many respects, P-type T4SS appear to be capable of transferring/secreting/taking up a
broader repertoire of macromolecules. For example, IncP
and IncI plasmids are also known to transfer the DNA
primases, TraC and Sog, respectively, from donor to recipient cells [21] even in the absence of DNA [22]. As
noted earlier, Helicobacter pylori utilizes a subset of the
P-type T4SS for DNA uptake [5]. Many pathogens use
P-type T4SS to secrete virulence factors into hosts as proteins or nucleoprotein complexes, such as the T-DNA of
the Ti plasmid [87], CagA of H. pylori [88^90] and pertussis toxin of B. pertussis [91,92]. It is noteworthy that conjugative plasmids containing the P-type T4SS are broadhost-range (IncP, W and N) [93] whereas F/H-type systems are narrow-host-range.
4. The nature of the conjugative pore
The nature of the conjugative pore is the central question in conjugation, as well as in the biology of T4SS.
Only recently have we begun to understand how singlestranded DNA can traverse the cell envelopes of both
donor and recipient cells (Fig. 2). At the inner face of
the conjugative pore are coupling proteins, which are
present in all conjugative transfer systems [19]. Coupling
proteins are inner membrane proteins that are thought to
recruit the cytoplasmic relaxosome complex to the membrane-associated T4SS [94,95] with direct interactions between relaxosomes and coupling proteins having recently
6
Fig. 4. Alignment of a portion of TraGF homologs to illustrate the evolutionary relationship between the N-terminal region of TraGF and members of
the VirB6 family. A PSI-BLAST search was performed as described in Fig. 3. Each sequence is labeled as follows: three-letter bacterial species abbreviation, plasmid name (if applicable), gene or protein name. TraG from plasmid F is outlined by a black box. Only the amino acid sequence extending
from L298 to K386 of the entire 938-amino acid sequence of TraGF is shown. The following list comprises all of the relevant information in the alignments in the following format: three-letter bacterial species abbreviation, plasmid name (if applicable), gene or protein name, accession number, full
bacterial species name. Atu pAT AvhB6 gi16119391 A. tumefaciens; Atu pAT VirB6 gi17938753 A. tumefaciens ; Ret VirB6 gi21492814 Rhizobium etli;
Sme pSymA VirB6 gi16263167 Sinorhizobium meliloti ; Lpn LvhB6 gi19919314 L. pneumophila; Lpn LvhB6 gi6249468 L. pneumophila; Xax pXA64
VirB6 gi21264269 Xanthomonas axonopodis; Ppu pWWo MpfE gi18150987 Pseudomonas putida; pIPO2T TraH gi16751940 Broad host range; Sme
pSB102 TraH gi15919984 S. meliloti ; Xca VirB6 gi21232730 Xanthomonas campestris pv. campestris; Xca VirB6 gi21232726 X. campestris pv. campestris ; Xca VirB6 gi21232559 X. campestris pv. campestris; Xca VirB6 gi21232723 X. campestris pv. campestris; Xax VirB6 gi21243338 X. axonopodis ;
Xax VirB6 gi21243338 X. axonopodis; Rrh pRi1724 riorf158 gi10954804 Rhizobium rhizogenes ; Atu pTiAB2 VirB6 gi18033167 A. tumefaciens; Atu pTi
VirB6 gi17939305 A. tumefaciens ; Atu pTiC58 VirB6 gi73223 A. tumefaciens; Atu pRiA VirB6 gi3184197 A. tumefaciens; Atu pTi VirB6 gi10955148
A. tumefaciens; Hpy jhp0033 gi15611104 H. pylori; Hpy orf1 gi4185987 H. pylori ; Rrh pRi1724 riorf124 gi10954770 R. rhizogenes; Atu pRiA4b TrbL
gi13990978 A. tumefaciens ; Rrh pNGR234a TrbL gi16519692 R. rhizogenes ; Atu pTi AGR_pTi_76p gi16119847 A. tumefaciens; Atu pTiSakura tiorf11
gi10954831 A. tumefaciens; Atu pTi TrbL gi10955098 A. tumefaciens ; Psp pADP1 TrbL gi13937498 Pseudomonas sp. ADP ; Cte pTSA TrbL
gi13661668 Comamonas testosteroni; Eae R751 TrbL gi10955221 Enterobacter aerogenes; Psp pB4 TrbL gi19352395 Pseudomonas sp.; RK2 TrbL
gi348633 Broad host range; Xfa pXF51 Xfa0037 gi10956748 Xylella fastidiosa; Mlo mlr6402 gi13475356 Mesorhizobium loti; Mlo TrbL gi20803852
M. loti ; Mlo pMLb m119606 gi13488455 M. loti ; Rso TrbL gi17547297 Ralstonia solanacearum; Sen pHCM1 HCM1.262 gi18466639 Salmonella enterica; Sty R27 TrhG gi10957317 S. typhi ; S£ R100 TraG gi9507645 Shigella £exneri ; Eco F TraG gi9507812 E. coli; Sty pSLT TraU gi17233465 S. typhimurium; Sty pED208 TraG gi21632647 S. typhi; Vco SXT TraG gi21885271 V. cholerae ; Pre R391 TraG gi20095130 P. rettgeri; Rco RC0146
gi15892069 Rickettsia canorii ; Rpr RP108 gi15603985 Rickettsia prowazekii ; Eco R388 TrwI gi2661722 E. coli; Sen SO11 gi12719015 S. enterica; Eco
R6K PilX6 gi12053572 E. coli ; Ype pYC orf5 gi10955839 Yersinia pestis; Sty R46 TraD gi17530595 S. typhimurium; Xfa pXF51 Xfa0011 gi10956722
X. fastidiosa; Mlo pMLa mlr9255 gi13488265 M. loti ; Mlo R71 msi411 gi20804241 M. loti; Ngo TraG gi14860859 N. gonorrhoeae; Pvu Rts1 orf242
gi21233904 P. vulgaris ; Bhe VirB6 gi6007533 Bartonella henselae ; Cac CAC2046 gi15895316 Clostridium acetobutylicum ; Nar pNL1 TraG gi10956931
N. aromaticivorans ; Eco R721 TraA gi10955522 E. coli; Ccr CC2420 gi16126659 Caulobacter crescentus; Bpe VirB6 gi420952 B. pertussis ; Hpy jhp0937
gi15612002 H. pylori; Sco SCD72A.15c gi21222528 Streptomyces coelicolor ; Ban pX01 pX01-79 gi10956326 Bacillus anthracis; Bf1 hydrophobic protein
gi1813499 Bacillus ¢rmus; Cac CAC2403 gi15895669 C. acetobutylicum; Bth TraJ gi10444274 Bacteroides thetaiotaomicron; Nsp alr7534 gi17158670 Nostoc sp. PCC 7120.
FEMSLE 11051 27-6-03
10
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
PreR391TraB VcoSXTTraB
SmeHtdB
100
StyR27TrhB
PvuRts1TraB
100
StypED208TraB
StyR100TraB
EcoFTraB
StypSLTTraB
98
100
100
RcoVirB10
100
EchVirB10
73
WolbachiaVirB10
64
NarpNL1TraB
89
88
MloR7ATrbI
89
Ccrcc2422
65
91
95
R64TraO
100
ReuTrbI
RsoTrbI
65
58
MlopMLbmll9603
100
65
97
LpnIcmE/DotG
XfapXF51Xfa40
LpnLvhB10
65
100
CtepTSATrbI
EaeR751TrbI
100
73
CcrVirB10
RP4TrbI
MlopMLamlr9259
98
AtupTiA6VirB10
RetVirB10
AtupOctopineVirB10
AtupTiC58VirB10
RrhpRi1724VirB10
BheVirB10
75
AtupATAGRpAT231
67
95
SmeVirB10
99
Retp42dVirB10
R388TrwE
XcaVirB10
65
86
65
100
AtupOctopineTrbI
100
68
100
65
100
RrhpRi1724TrbI
XaxpXAC64VirB10
RrhpNGR234aTrbI
65
PpupWWompfH
65
StyR46TraF
59
65
100
XaxVirB10
65
65
65
AacVirB10
85
65
BpeVirB10
HinaegyptiuspBF3031VirB10
100
88
HpyJ99orf1314
Hpyhp0527
EcoPilX10
Xfaxfa0014
BmeAbortusVirB10
EcoR721TraI
100
CjeComB3
HpyorfJ
HpyComB3
50 changes
been demonstrated [14,15]. The hexameric coupling protein is anchored in the inner membrane with the cytoplasY in
mic domain forming a channel that measures 22 A
diameter, which could easily accommodate a single strand
Y ). The coupling protein is thought to use
of DNA (V10 A
ATP hydrolysis to energize the ‘pumping’ of DNA
through the coupling protein channel [19,96].
Recently, the coupling protein of R27, TraGH , an
FEMSLE 11051 27-6-03
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
F-type system, has been shown to interact with the
N-terminus of TrhBH , a member of the TraBF family.
TrhBH was also shown to form multimers, possibly forming a ring structure that could extend the pore of the
coupling protein into the periplasmic space [59]. TraBF
of the F factor also interacts with TraKF , which in turn
interacts with TraVF , a lipoprotein that could stabilize the
secretin-like TraKF protein [49]. Secretins are known to
form gated, outer membrane rings that allow the passage
of macromolecules in response to a signal that opens the
pore [97,98]. A TraKF secretin-like structure, anchored by
TraVF , could, therefore, extend the conjugative pore from
the coupling protein through to the outer membrane, via
TraBF . Although there is evidence for such a structure in
other secretion systems, this needs to be demonstrated experimentally for the T4SS.
Consistent with the idea of TraBF , -KF and -VF forming
the core of the pore which transfers DNA, expression of
VirB3Ti , -B4Ti , -B7Ti , -B8Ti , -B9Ti and -B10Ti of the Ti
plasmid in recipient cells increases the e⁄ciency of
RSF1010 transfer [99]. This suggests that the presence of
these proteins within recipients aids in the transport of the
DNA into the cytoplasm. Since all of these VirB proteins,
except VirB8Ti , have a homolog/analog in F-like T4SS,
including the sca¡olding proteins of the putative pore
(TraBF , -KF and -VF ; Table 1), it is likely that the pore
extends from the donor inner membrane to the recipient
cytoplasm. Consistent with this proposal, homologs of
VirB7Ti and -B10Ti have been shown to be responsible
for DNA uptake by H. pylori [5], illustrating that these
proteins likely represent the minimal membrane-spanning
pore for DNA transfer.
Although these observations suggest a mechanism by
which DNA could cross the donor envelope, the mechanism by which the DNA traverses the recipient envelope
to gain access to the cytoplasm remains a key question.
Some evidence is available that suggests the T4SS system
11
of F penetrates the recipient cell. TraGF has been implicated in entry exclusion involving protein^protein interactions between TraGF and TraSF , the entry exclusion protein, located in the donor and recipient cells, respectively
[32]. This suggests that TraGF is translocated into the
recipient cell and interacts with TraSF to block DNA
transfer. Also, 35 S-labelled TraNF and possibly TraUF
are found in the recipient cell after separation of the donor
and recipient cells using magnetic bead technology (L.S.
Frost, unpublished results). Since the net outcome of F-,
P- and I-type conjugative systems is the same, there must
be an underlying mechanism common to all T4SS, which
do di¡er somewhat in their repertoire of proteins that
promote pilus assembly and DNA transport. Does the
F-pilus retract, and if it does, do the P- and I-type pili
also retract ? Do P-type systems also translocate proteins
into the recipient cell to form a stable mating junction?
Does the DNA transfer through the pilus, situated within
the conjugative pore, with the pilus penetrating the recipient cell envelope and depositing the DNA directly within
the recipient cytoplasm, much like a phage tail tube within
the contractile tails of T-even phages injects DNA? The
idea that pili can be used to transport macromolecules is
supported by the ¢ndings of Jin and He [100,101], who
visualized protein secretion from the tips of type III secretion system pili. This observation implies that pili can indeed serve as a conduit for macromolecular tra⁄cking.
5. Relationships between T4SS, T3SS and T2SS
Gram-negative bacteria possess multiple pathways for
secreting macromolecules across the outer membrane
[102], with conjugation via T4SS being one of the more
complex pathways [8]. Secretion pathways with interesting
similarities to T4SS are the type II secretion systems
(T2SS; 12^16 proteins) and the type III secretion systems
6
Fig. 5. A phylogenetic tree illustrating the evolutionary relationship between TraBF , TrbIP and VirB10Ti . The tree was constructed as described in Fig.
3, and labeled using the same style of annotations. The following list comprises all of the relevant information in the homology tree in the following
format: three-letter bacterial species abbreviation, plasmid name, gene name, accession number, full bacterial species name. Pvu RTS1 orf210
NP_640170 P. vulgaris ; Vco SXT TraB AAL59682 V. cholerae ; Pre R391 TraB AAM08009 P. rettgeri; Sme R478 HtdB AAD01913 S. marcescens; Sty
R27 TrhB AAD54048 S. typhi; Sty pED208 TraB AAM90707 S. typhi; Sty R100 TraB BAA78855 S. typhimurium ; Eco F TraB AAC44179 E. coli ;
Sty pSLT TraB AAL23486 S. typhimurium ; Nar pNL1 TraB NP_049165 N. aromaticivorans; Sty R64 TraO BAA78003 S. typhimurium ; Lpn IcmE
CAA75165 L. pneumophila; Lpn LvhB10 CAB60060 L. pneumophila ; Ccr CC2422 NP_421225 C. crescentus ; Mlo pMLa mlr9259 NP_085799 M. loti;
Ret pa VirB10 AAD55069 R. etli; Atu pTiA6 VirB10 AAF77170 A. tumefaciens ; Atu octopine-like Ti VirB10 NP_059808 A. tumefaciens; Atu pTiC58
VirB10 P17800 A. tumefaciens; Rrh pRi1724 riorf162 NP_066743 R. rhizogenes ; Bhe VirB10 AAF00948 B. henselae; Atu pAT AGR_pAT_231p
NP_396101 A. tumefaciens ; Sme pSymA VirB10 NP_435956 S. meliloti ; Ret p42d VirB10 NP_659885 R. etli; Eco R388 TrwE CAA57031 E. coli; Xca
VirB10 NP_637831 X. campestris ; Xax VirB10 NP_642932 X. axonopodis; Bpe VirB10 E47301 B. pertussis ; Hpy orf13/14 NP_223194 H. pylori strain
J99; Hpy HP0527 AAD07594 H. pylori strain 26695; Hpy orfJ AAM03036 H. pylori strain PeCan18B ; Hpy ComB3 CAA10657 H. pylori strain P1;
Cje pVir VirB10 AAF97747 Campylobacter jejuni; Eco R6K PilX10 CAC20148 E. coli; Bme VirB10 AAF73903 Brucella melitensis bv. abortus; Xfa
pXF51 XFa0014 AAF85583 X. fastidiosa; Eco R721 TraI NP_065358 E. coli; Hin pBF3028 Bp120 NP_660236 Haemophilus in£uenzae biotype aegyptius; Aac pVT745 magB10 NP_067574 Actinobacillus actinomycetemcomitans; Sty R46 TraF NP_511197 S. typhimurium; Ppu pWWo MpfH NP_542921
P. putida; Xax pXAC64 VirB10 AAM39284 X. axonopodis; Rrh pNGR234a TrbI NP_443816 R. rhizogenes ; Rrh pRi1724 riorf120 NP_066701 R. rhizogenes ; Atu octopine-like Ti TrbI NP_059750 A. tumefaciens; Eco RP4 TrbI AAA26435 E. coli; Eae R751 TrbI AAC64450 E. aerogenes; Cte pTSA
TrbI AAK38009 C. testosteroni; Xfa pXF51 XFa0040 AAF85609 X. fastidiosa; Mlo pMLb mll9603 NP_109459 M. loti; Rso TrbI NP_520696 R. solanacearum ; Reu Tn4371 TrbI CAA71794 Ralstonia eutropha; Ccr CC2685 AAK24651 C. crescentus; Mlo TrbI CAD31427 M. loti ; Wol VirB10
BAA97441 Wolbachia sp. strain wKueYO ; Ech VirB10 AAM00413 Ehrlichia cha¡eensis; Rco VirB10 AAL02927 R. canorii.
FEMSLE 11051 27-6-03
12
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
(T3SS; 20 proteins). T2SS are one of the terminal
branches of the general secretory pathway, which is responsible for secreting a wide range of extracellular toxins
and enzymes by Gram-negative bacteria [103]. T2SS are
also closely related to secretion pathways for the biosynthesis of type IV pili [104]. Among the T3SS are molecular
syringes that inject virulence e¡ector proteins directly into
the cytoplasm of host cell [105]. T3SS also share both sequence and structural similarities with £agellar basal
bodies [106,107].
Based on in silico analysis, the homology between T4SS,
T3SS and T2SS is quite limited. However, each system
does contain a secretin protein and an associated stabilizing lipoprotein, which together could function as a gated
outer membrane channel that allows the passage of macromolecules. Each system also contains one or two
NTPases that likely energize either assembly of the secretion apparatus or macromolecule secretion. NTPases contained within the T2SS (GspE) are homologous to the
NTPases from P- and I-type T4SS (i.e. TrbBP /VirB11Ti
and TraJI ), but not NTPases from F-like T4SS [26].
How energy is utilized in these systems will be key to
understanding their di¡erences. Structural determination
of key transfer proteins will undoubtedly provide valuable
insight into the nature of T4SS that cannot be obtained
from database searches. For example, the crystal structure
of the coupling protein TrwB identi¢ed structural homologies to DNA ring helicases and therefore suggested a
mechanism by which single-stranded DNA could be actively pumped through the conjugative pore. It will be
interesting to determine if any homology exists between
T4SS, T2SS and T3SS at the level of protein structure
and whether the theme of interacting proteins assembled
into multimeric rings is common to many secretion systems.
From a mechanistic and anatomical standpoint, there
are striking similarities between T4SS, T3SS and T2SS.
Each secretion system is a multi-protein, membrane-associated complex that can assemble ¢lamentous appendages,
such as pili or £agella, on the bacterial cell surface and are
involved in macromolecular transport. Many type II and
IV systems share the properties of retractile pili [12,76] and
sensitivity to pilus-speci¢c bacteriophages [75], which presumably take advantage of pilus retraction for entry into
the host. Some type III and IV systems share an ability to
trigger macromolecular transport in response to contact
with host eukaryotic cells [108] or bacterial cells [68], respectively. Although the molecular mechanisms for each
of these processes are not yet fully understood, various
aspects of these secretion pathways appear to be conserved, possibly re£ecting a common evolutionary origin
of either complete systems or modular components of each
system.
Interesting parallels exist between the substrates secreted
by T4SS and T2SS. For example, natural transformation,
or DNA uptake, can be mediated by either T2SS [109] or
T4SS [5]. Also, secretion of structurally similar toxins can
occur by either a T2SS (cholera toxin) [103] or a T4SS
(pertussis toxin) [91,92]. Another interesting comparison
involves DNA transfer mediated by the F T4SS that
shares mechanistic similarities to ¢lamentous phage
(M13 and f1) replication and packaging, which uses a
secretin/lipoprotein channel, thioredoxin and an NTPase
[104]. Both systems use an evolutionarily related mechanism to produce a single-stranded DNA intermediate via
rolling circle replication [110,111], which is either transferred to a recipient or packaged upon phage extrusion.
The T4SS gene products assemble the conjugative pilus, a
structure that is structurally related to class I ¢lamentous
phages, which consists of a helical array of proteins
around a circular, single-stranded DNA molecule [112],
possibly providing insight into the transport of DNA during conjugation.
The secretion system classi¢cation scheme (T2^T4SS)
conveniently divides important pathways into logical categories, which has greatly facilitated the study and understanding of these systems [102]. However, the expanding
genome databases and the molecular dissection of several
model secretion systems has revealed both the diversity
within and the shared relationships between secretion system categories. From an evolutionary perspective, these
observations make it tempting to speculate that numerous
variations of secretion pathways exist that are built on a
¢nite array of central modular components.
6. Future studies on T4SS
Identi¢cation by genetic and computer-based methods
of the essential components of T4SS provides a foundation to ask more detailed questions about the mechanism
of macromolecular secretion, in general. Careful biochemical and genetic analysis of individual transfer proteins will
continue to provide valuable insight into the mechanics of
secretion. Methods to determine protein^protein interactions will be central to constructing a detailed model of the
T4SS apparatus since microscopic analyses, so far, have
proven uninformative. Such examples include the identi¢cation of the TraBF , -KF , -VF envelope-spanning structure
[49] and identi¢cation of an interaction between TrhBH
and the coupling protein TraGH [47] and the many examples in the VirBTi literature [2]. Identifying how macromolecules access the pore and initiate the transfer process
as well as their e¡ect on the recipient as they enter the
cytoplasm will also be key questions [10].
Bacterial conjugation provides a model system for
studying bacterial signaling as the nature of the ever elusive mating signal remains unknown. It is anticipated that
an external cue, possibly involving contact between donor
and recipient, is transferred via the pilus, through the
membrane-associated T4SS and coupling protein to the
cytoplasmic relaxosome. This process appears to involve
FEMSLE 11051 27-6-03
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
pilus retraction, an as yet poorly understood phenomenon.
Several T4SS components contain features of signaling
molecules. For example, coiled-coil domains, such as those
in TraBF and TraHF , which can undergo modi¢cation,
have been implicated in molecular signaling [113] and
modulation of binding through changes in the local cellular environment [114]. This signal could then trigger events
that resemble phage infection and injection of DNA or the
injection of proteins in a contact-mediated manner as seen
in T3SS.
[14]
[15]
[16]
[17]
Acknowledgements
[18]
The authors wish to thank Bart Hazes, Diane Taylor
and members of her lab for unpublished data. We also
wish to thank Sean Graham for his help in generating
the phylogenetic tree data.
[19]
[20]
[21]
References
[22]
[1] Lederberg, J. and Tatum, E.L. (1946) Gene recombination in Escherichia coli. Nature 158, 558.
[2] Christie, P.J. (2001) Type IV secretion : intercellular transfer of macromolecules by systems ancestrally related to conjugation machines.
Mol. Microbiol. 40, 294^305.
[3] Taylor, D.E., Gibreel, A., Lawley, T.D. and Tracz, D.M. (2003)
Antibiotic resistance plasmids. In: Biology of Plasmids (Philips, G.
and Funnel, B., Eds.), in press. ASM Press, Washington, DC.
[4] Christie, P.J. and Vogel, J.P. (2000) Bacterial type IV secretion: conjugation systems adapted to deliver e¡ector molecules to host cells.
Trends Microbiol. 8, 354^360.
[5] Hofreuter, D., Odenbreit, S. and Haas, R. (2001) Natural transformation competence in Helicobacter pylori is mediated by the basic
components of a type IV secretion system. Mol. Microbiol. 41, 379^
391.
[6] Firth, N., Ippen-Ihler, K. and Skurray, R.A. (1996) Structure and
function of the F-factor and mechanism of conjugation. In: Escherichia coli and Salmonella : Cellular and Molecular Biology, Vol. 1
(Neidhardt, F.C., Ed.), pp. 2377^2401. ASM Press, Washington, DC.
[7] Frost, L.S., Ippen-Ihler, K. and Skurray, R.A. (1994) Analysis of the
sequence and gene products of the transfer region of the F sex factor.
Microbiol. Rev. 58, 162^210.
[8] Lawley, T.D., Wilkins, B.M. and Frost, L.S. (2003) Bacterial conjugation in gram-negative bacteria. In: Biology of Plasmids (Phillips,
G. and Funnel, B., Eds.), in press. ASM Press, Washington, DC.
[9] Durrenberger, M.B., Villiger, W. and Bachi, T. (1991) Conjugational
junctions : morphology of speci¢c contacts in conjugating Escherichia
coli bacteria. J. Struct. Biol. 107, 146^156.
[10] Lawley, T.D., Gordon, G.S., Wright, A. and Taylor, D.E. (2002)
Bacterial conjugative transfer: visualization of successful mating pairs
and plasmid establishment in live Escherichia coli. Mol. Microbiol.
44, 947^956.
[11] Samuels, A.L., Lanka, E. and Davies, J.E. (2000) Conjugative junctions in RP4-mediated mating of Escherichia coli. J. Bacteriol. 182,
2709^2715.
[12] Novotny, C.P. and Fives-Taylor, P. (1974) Retraction of F pili.
J. Bacteriol. 117, 1306^1311.
[13] Clewell, D.B. and Helinski, D.R. (1969) Supercoiled circular DNAprotein complex in Escherichia coli : puri¢cation and induced conver-
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
13
sion to an open circular DNA form. Proc. Natl. Acad. Sci. USA 62,
1159^1166.
Schroder, G., Krause, S., Zechner, E.L., Traxler, B., Yeo, H.J., Lurz,
R., Waksman, G. and Lanka, E. (2002) TraG-like proteins of DNA
transfer systems and of the Helicobacter pylori type IV secretion
system : inner membrane gate for exported substrates? J. Bacteriol.
184, 2767^2779.
Szpirer, C.Y., Faelen, M. and Couturier, M. (2000) Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization
protein Mob. Mol. Microbiol. 37, 1283^1292.
Ohki, M. and Tomizawa, J.I. (1968) Asymmetric transfer of DNA
strands in bacterial conjugation. Cold Spring Harbor Symp. Quant.
Biol. 33, 651^658.
Rupp, W.D. and Ihler, G. (1968) Strand selection during bacterial
mating. Cold Spring Harbor Symp. Quant. Biol. 33, 647^650.
Zechner, E.L. et al. (2000) Conjugative-DNA transfer processes. In:
The Horizontal Gene Pool (Thomas, C.M., Ed.), pp. 87^174. Harwood Academic, Amsterdam.
Llosa, M., Gomis-Ruth, F.X., Coll, M. and de la Cruz, F. (2002)
Bacterial conjugation : a two-step mechanism for DNA transport.
Mol. Microbiol. 45, 1^8.
Henderson, D. and Meyer, R. (1999) The MobA-linked primase is
the only replication protein of R1162 required for conjugal mobilization. J. Bacteriol. 181, 2973^2978.
Rees, C.E. and Wilkins, B.M. (1990) Protein transfer into the recipient cell during bacterial conjugation : studies with F and RP4. Mol.
Microbiol. 4, 1199^1205.
Wilkins, B.M. and Thomas, A.T. (2000) DNA-independent transport
of plasmid primase protein between bacteria by the I1 conjugation
system. Mol. Microbiol. 38, 650^657.
Herrera-Estrella, A., Van Montagau, M. and Wang, K. (1990) A
bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a betagalactosidase fusion protein into tobacco nuclei. Proc. Natl. Acad.
Sci. USA 87, 9534^9537.
Howard, E.A., Zupan, J.R., Citovsky, V. and Zambryski, P.C. (1992)
The VirD2 protein of A. tumefaciens contains a C-terminal bipartite
nuclear localization signal implications for nuclear uptake of DNA in
plant cells. Cell 68, 109^118.
Ziemienowicz, A., Merkle, T., Schoumacher, F., Hohn, B. and Rossi,
L. (2001) Import of Agrobacterium T-DNA into plant nuclei. Two
distinct functions of VirD2 and VirE2 proteins. Plant Cell 13, 369^
384.
Planet, P.J., Kachlany, S.C., DeSalle, R. and Figurski, D.H. (2001)
Phylogeny of genes for secretion NTPases: identi¢cation of the widespread tadA subfamily and development of a diagnostic key for gene
classi¢cation. Proc. Natl. Acad. Sci. USA 98, 2503^2508.
Bradley, D.E., Taylor, D.E. and Cohen, D.R. (1980) Speci¢cation of
surface mating systems among conjugative drug resistance plasmids
in Escherichia coli K-12. J. Bacteriol. 143, 1466^1470.
Lanka, E. and Wilkins, B.M. (1995) DNA processing reactions in
bacterial conjugation. Annu. Rev. Biochem. 64, 141^169.
Komano, T., Yoshida, T., Narahara, K. and Furuya, N. (2000) The
transfer region of IncI1 plasmid R64: similarities between R64 tra
and Legionella icm/dot genes. Mol. Microbiol. 35, 1348^1359.
Segal, G., Purcell, M. and Shuman, H.A. (1998) 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 95, 1669^1674.
Vogel, J.P., Andrews, H.L., Wong, S.K. and Isberg, R.R. (1998)
Conjugative transfer by the virulence system of Legionella pneumophila. Science 279, 873^876.
Anthony, K.G., Klimke, W.A., Manchak, J. and Frost, L.S. (1999)
Comparison of proteins involved in pilus synthesis and mating pair
stabilization from the related plasmids F and R100-1: insights into
the mechanism of conjugation. J. Bacteriol. 181, 5149^5159.
Lawley, T.D., Gilmour, M.W., Gunton, J.E., Standeven, L.J. and
FEMSLE 11051 27-6-03
14
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
Taylor, D.E. (2002) Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27. J. Bacteriol. 184, 2173^2180.
Lawley, T.D., Gilmour, M.W., Gunton, J.E., Tracz, D.M. and Taylor, D.E. (2003) Functional and mutational analysis of the conjugative transfer region 2 (Tra2) of the IncHI1 plasmid R27. J. Bacteriol.
185, 581^591.
Majdalani, N., Moore, D., Maneewannakul, S. and Ippen-Ihler, K.
(1996) Role of the propilin leader peptide in the maturation of F pilin.
J. Bacteriol. 178, 3748^3754.
Majdalani, N. and Ippen-Ihler, K. (1996) Membrane insertion of the
F-pilin subunit is Sec independent but requires leader peptidase B
and the proton motive force. J. Bacteriol. 178, 3742^3747.
Harris, R.L., Sholl, K.A., Conrad, M.N., Dresser, M.E. and Silverman, P.M. (1999) Interaction between the F plasmid TraA (F-pilin)
and TraQ proteins. Mol. Microbiol. 34, 780^791.
Manchak, J., Anthony, K.G. and Frost, L.S. (2002) Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection
and DNA transfer. Mol. Microbiol. 43, 195^205.
Lu, J., Manchak, J., Klimke, W., Davidson, C., Firth, N., Skurray,
R. and Frost, L. (2002) Analysis and characterization of the IncFV
plasmid pED208 transfer region. Plasmid 48, 24^37.
Frost, L.S., Finlay, B.B., Opgenorth, A., Paranchych, W. and Lee,
J.S. (1985) Characterization and sequence analysis of pilin from
F-like plasmids. J. Bacteriol. 164, 1238^1247.
Maneewannakul, K., Maneewannakul, S. and Ippen-Ihler, K. (1993)
Synthesis of F pilin. J. Bacteriol. 175, 1384^1391.
Eisenbrandt, R., Kalkum, M., Lai, E.M., Lurz, R., Kado, C.I. and
Lanka, E. (1999) Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits. J. Biol. Chem. 274,
22548^22555.
Moore, D., Sowa, B.A. and Ippen-Ihler, K. (1981) Location of an
F-pilin pool in the inner membrane. J. Bacteriol. 146, 251^259.
Maher, D., Sherburne, R. and Taylor, D.E. (1993) H-pilus assembly
kinetics determined by electron microscopy. J. Bacteriol. 175, 2175^
2183.
Shirasu, K. and Kado, C.I. (1993) Membrane location of the Ti
plasmid VirB proteins involved in the biosynthesis of a pilin-like
conjugative structure on Agrobacterium tumefaciens. FEMS Microbiol. Lett. 111, 287^294.
Grahn, A.M., Haase, J., Bamford, D.H. and Lanka, E. (2000) Components of the RP4 conjugative transfer apparatus form an envelope
structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems. J. Bacteriol.
182, 1564^1574.
Gilmour, M.W., Lawley, T.D., Rooker, M.M., Newnham, P.J. and
Taylor, D.E. (2001) Cellular location and temperature-dependent assembly of IncHI1 plasmid R27-encoded TrhC-associated conjugative
transfer protein complexes. Mol. Microbiol. 42, 705^715.
Schmidt-Eisenlohr, H., Domke, N., Angerer, C., Wanner, G., Zambryski, P.C. and Baron, C. (1999) Vir proteins stabilize VirB5 and
mediate its association with the T pilus of Agrobacterium tumefaciens.
J. Bacteriol. 181, 7485^7492.
Harris, R.L., Hombs, V. and Silverman, P.M. (2001) Evidence that
F-plasmid proteins TraV, TraK and TraB assemble into an envelopespanning structure in Escherichia coli. Mol. Microbiol. 42, 757^
766.
Deng, W.L. and Huang, H.C. (1999) Cellular locations of Pseudomonas syringae pv. syringae HrcC and HrcJ proteins, required for harpin
secretion via the type III pathway. J. Bacteriol. 181, 2298^2301.
Guilvout, I., Hardie, K.R., Sauvonnet, N. and Pugsley, A.P. (1999)
Genetic dissection of the outer membrane secretin PulD: are there
distinct domains for multimerization and secretion speci¢city? J. Bacteriol. 181, 7212^7220.
Baron, C., O‘Callaghan, D. and Lanka, E. (2002) Bacterial secrets of
secretion : EuroConference on the biology of type IV secretion processes. Mol. Microbiol. 43, 1359^1365.
[53] Schandel, K.A., Muller, M.M. and Webster, R.E. (1992) Localization
of TraC, a protein involved in assembly of the F conjugative pilus.
J. Bacteriol. 174, 3800^3806.
[54] Cao, T.B. and Saier Jr., M.H. (2001) Conjugal type IV macromolecular transfer systems of Gram-negative bacteria : organismal distribution, structural constraints and evolutionary conclusions. Microbiology 147, 3201^3214.
[55] Rabel, C., Grahn, A.M., Lurz, R. and Lanka, E. (2003) The VirB4
family of proposed tra⁄c nucleoside triphosphatases : common motifs in plasmid RP4 TrbE are essential for conjugation and phage
adsorption. J. Bacteriol. 185, 1045^1058.
[56] Schandel, K.A., Maneewannakul, S., Ippen-Ihler, K. and Webster,
R.E. (1987) A traC mutant that retains sensitivity to f1 bacteriophage
but lacks F pili. J. Bacteriol. 169, 3151^3159.
[57] Firth, N. and Skurray, R. (1992) Characterization of the F plasmid
bifunctional conjugation gene, traG. Mol. Gen. Genet. 232, 145^
153.
[58] Jones, C.J., Homma, M. and Macnab, R.M. (1989) L-, P-, and
M-ring proteins of the £agellar basal body of Salmonella typhimurium: gene sequences and deduced protein sequences. J. Bacteriol. 171,
3890^3900.
[59] Gilmour, M.W., Gunton, J.E., Lawley, T.D. and Taylor, D.E. (2003)
Interaction between the IncHI1 plasmid R27 coupling protein and a
component of the R27 Type IV secretion system : TraG associates
with the coiled-coil mating pair formation protein TrhB. Mol. Microbiol. (in press).
[60] Bliska, J. (1996) How pathogens exploit interactions mediated by
SH3 domains. J. Chem. Biol. 3, 7^11.
[61] Klimke, W. (2002) PhD Thesis, Analysis of the mating pair stabilization system of the F-plasmid. Department of Biological Sciences,
University of Alberta, Edmonton, AB.
[62] Collet, J.F. and Bardwell, J.C. (2002) Oxidative protein folding in
bacteria. Mol. Microbiol. 44, 1^8.
[63] Manwaring, N. (2001) PhD Thesis, Molecular analysis of the conjugative F-plasmid leading and transfer regions. School of Biological
Sciences University of Sydney, Sydney, NSW.
[64] Maneewannakul, S., Kathir, P. and Ippen-Ihler, K. (1992) Characterization of the F plasmid mating aggregation gene traN and of a new
F transfer region locus trbE. J. Mol. Biol. 225, 299^311.
[65] Harris, R.L. and Silverman, P.M. (2002) Roles of internal cysteines
in the function, localization, and reactivity of the TraV outer membrane lipoprotein encoded by the F plasmid. J. Bacteriol. 184, 3126^
3129.
[66] Baron, C., Thorstenson, Y.R. and Zambryski, P. (1997) The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium
tumefaciens. J. Bacteriol. 179, 1211^1218.
[67] Das, A., Anderson, L.B. and Xie, Y.H. (1997) Delineation of the
interaction domains of Agrobacterium tumefaciens VirB7 and VirB9
by use of the yeast two-hybrid assay. J. Bacteriol. 179, 3404^3409.
[68] Kingsman, A. and Willetts, N. (1978) The requirements for conjugal
DNA synthesis in the donor strain during Flac transfer. J. Mol. Biol.
122, 287^300.
[69] Achtman, M., Kennedy, N. and Skurray, R. (1977) Cell-cell interactions in conjugating Escherichia coli: role of TraT protein in surface
exclusion. Proc. Natl. Acad. Sci. USA 74, 5104^5108.
[70] Klimke, W.A., Kennedy, R.A., Rypien, C.D., Harris, R.L., Silverman, P.M. and Frost, L.S. (2003) Analysis of the mating pair stabilization protein, TraN, of the F plasmid, reveals a dependence on
disul¢de bond formation for stability and function. J. Bacteriol. (submitted).
[71] Klimke, W.A. and Frost, L.S. (1998) Genetic analysis of the role of
the transfer gene, traN, of the F and R100-1 plasmids in mating pair
stabilization during conjugation. J. Bacteriol. 180, 4036^4043.
[72] Moore, D., Maneewannakul, K., Maneewannakul, S., Wu, J.H., Ippen-Ihler, K. and Bradley, D.E. (1990) Characterization of the
F-plasmid conjugative transfer gene traU. J. Bacteriol. 172, 4263^
4270.
FEMSLE 11051 27-6-03
T.D. Lawley et al. / FEMS Microbiology Letters 224 (2003) 1^15
[73] Bradley, D.E. (1980) Morphological and serological relationships of
conjugative pili. Plasmid 4, 155^169.
[74] Novotny, C.P. and Fives-Taylor, P. (1978) E¡ects of high temperature on Escherichia coli F pili. J. Bacteriol. 133, 459^464.
[75] Paranchych, W. and Frost, L.S. (1988) The physiology and biochemistry of pili. Adv. Microb. Physiol. 29, 53^114.
[76] Merz, A.J., So, M. and Sheetz, M.P. (2000) Pilus retraction powers
bacterial twitching motility. Nature 407, 98^102.
[77] Skerker, J.M. and Berg, H.C. (2001) Direct observation of extension
and retraction of type IV pili. Proc. Natl. Acad. Sci. USA 98, 6901^
6904.
[78] Sherburne, C.K., Lawley, T.D., Gilmour, M.W., Blattner, F.R., Burland, V., Grotbeck, E., Rose, D.J. and Taylor, D.E. (2000) The
complete DNA sequence and analysis of R27, a large IncHI plasmid
from Salmonella typhi that is temperature sensitive for transfer. Nucleic Acids Res. 28, 2177^2186.
[79] Murata, T. et al. (2002) Complete nucleotide sequence of plasmid
Rts1: implications for evolution of large plasmid genomes. J. Bacteriol. 184, 3194^3202.
[80] Boltner, D., MacMahon, C., Pembroke, J.T., Strike, P. and Osborn,
A.M. (2002) R391: a conjugative integrating mosaic comprised of
phage, plasmid, and transposon elements. J. Bacteriol. 184, 5158^
5169.
[81] Beaber, J.W., Hochhut, B. and Waldor, M.K. (2002) Genomic and
functional analyses of SXT, an integrating antibiotic resistance gene
transfer element derived from Vibrio cholerae. J. Bacteriol. 184, 4259^
4269.
[82] Romine, M.F. et al. (1999) Complete sequence of a 184-kilobase
catabolic plasmid from Sphingomonas aromaticivorans F199. J. Bacteriol. 181, 1585^1602.
[83] Hamilton, H.L., Schwartz, K.J. and Dillard, J.P. (2001) Insertionduplication mutagenesis of Neisseria : use in characterization of
DNA transfer genes in the gonococcal genetic island. J. Bacteriol.
183, 4718^4726.
[84] Pansegrau, W. et al. (1994) Complete nucleotide sequence of Birmingham IncPK plasmids. Compilation and comparative analysis.
J. Mol. Biol. 239, 623^663.
[85] Thorsted, P.B. et al. (1998) Complete sequence of the IncPL plasmid
R751: implications for evolution and organisation of the IncP backbone. J. Mol. Biol. 282, 969^990.
[86] Winans, S.C. and Walker, G.C. (1985) Conjugal transfer system of
the IncN plasmid pKM101. J. Bacteriol. 161, 402^410.
[87] Zhu, J., Oger, P.M., Schrammeijer, B., Hooykaas, P.J., Farrand, S.K.
and Winans, S.C. (2000) The bases of crown gall tumorigenesis.
J. Bacteriol. 182, 3885^3895.
[88] Backert, S., Ziska, E., Brinkmann, V., Zimny-Arndt, U., Fauconnier,
A., Jungblut, P.R., Naumann, M. and Meyer, T.F. (2000) Translocation of the Helicobacter pylori CagA protein in gastric epithelial
cells by a type IV secretion apparatus. Cell. Microbiol. 2, 155^164.
[89] Segal, E.D., Cha, J., Lo, J., Falkow, S. and Tompkins, L.S. (1999)
Altered states: involvement of phosphorylated CagA in the induction
of host cellular growth changes by Helicobacter pylori. Proc. Natl.
Acad. Sci. USA 96, 14559^14564.
[90] Stein, M., Rappuoli, R. and Covacci, A. (2000) Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host
cell translocation. Proc. Natl. Acad. Sci. USA 97, 1263^1268.
[91] Covacci, A. and Rappuoli, R. (1993) Pertussis toxin export requires
accessory genes located downstream from the pertussis toxin operon.
Mol. Microbiol. 8, 429^434.
[92] Weiss, A.A., Johnson, F.D. and Burns, D.L. (1993) Molecular characterization of an operon required for pertussis toxin secretion. Proc.
Natl. Acad. Sci. USA 90, 2970^2974.
[93] Guiney, D.G. (1993) Broad host range conjugative and mobilizable
plasmids in gram-negative bacteriain: In: Bacterial Conjugation
(Thomas, C.M., Ed.), pp. 75^104. Plenum Press, New York.
15
[94] Cabezon, E., Sastre, J.I. and de la Cruz, F. (1997) Genetic evidence
of a coupling role for the TraG protein family in bacterial conjugation. Mol. Gen. Genet. 254, 400^406.
[95] Hamilton, C.M. et al. (2000) TraG from RP4 and TraG and VirD4
from Ti plasmids confer relaxosome speci¢city to the conjugal transfer system of pTiC58. J. Bacteriol. 182, 1541^1548.
[96] Panicker, M.M. and Minkley, E.G. (1985) DNA transfer occurs
during a cell surface contact stage of F sex factor-mediated bacterial
conjugation. J. Bacteriol. 162, 584^590.
[97] Nouwen, N., Stahlberg, H., Pugsley, A.P. and Engel, A. (2000)
Domain structure of secretin PulD revealed by limited proteolysis
and electron microscopy. EMBO J. 19, 2229^2236.
[98] Nouwen, N., Ranson, N., Saibil, H., Wolpensinger, B., Engel, A.,
Ghazi, A. and Pugsley, A.P. (1999) Secretin PulD: association with
pilot PulS, structure, and ion-conducting channel formation. Proc.
Natl. Acad. Sci. USA 96, 8173^8177.
[99] Bohne, J., Yim, A. and Binns, A.N. (1998) The Ti plasmid increases
the e⁄ciency of Agrobacterium tumefaciens as a recipient in VirBmediated conjugal transfer of an IncQ plasmid. Proc. Natl. Acad.
Sci. USA 95, 7057^7062.
[100] Jin, Q. and He, S.Y. (2001) Role of the Hrp pilus in type III protein
secretion in Pseudomonas syringae. Science 294, 2556^2558.
[101] Jin, Q., Hu, W., Brown, I., McGhee, G., Hart, P., Jones, A.L. and
He, S.Y. (2001) Visualization of secreted Hrp and Avr proteins
along the Hrp pilus during type III secretion in Erwinia amylovora
and Pseudomonas syringae. Mol. Microbiol. 40, 1129^1139.
[102] Thanassi, D.G. and Hultgren, S.J. (2000) Multiple pathways allow
protein secretion across the bacterial outer membrane. Curr. Opin.
Cell Biol. 12, 420^430.
[103] Sandkvist, M. (2001) Biology of type II secretion. Mol. Microbiol.
40, 271^283.
[104] Russel, M. (1998) Macromolecular assembly and secretion across
the bacterial cell envelope: type II protein secretion systems.
J. Mol. Biol. 279, 485^499.
[105] Buttner, D. and Bonas, U. (2002) Port of entry-the type III secretion
translocon. Trends Microbiol. 10, 186^192.
[106] Galan, J.E. and Collmer, A. (1999) Type III secretion machines :
bacterial devices for protein delivery into host cells. Science 284,
1322^1328.
[107] Kubori, T., Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tejero,
M., Sukhan, A., Galan, J.E. and Aizawa, S.I. (1998) Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280, 602^605.
[108] Galan, J.E. (2001) Salmonella interactions with host cells: type III
secretion at work. Annu. Rev. Cell Dev. Biol. 17, 53^86.
[109] Dubnau, D. (1999) DNA uptake in bacteria. Annu. Rev. Microbiol.
53, 217^244.
[110] Asano, S., Higashitani, A. and Horiuchi, K. (1999) Filamentous
phage replication initiator protein gpII forms a covalent complex
with the 5P end of the nick it introduced. Nucleic Acids Res. 27,
1882^1889.
[111] Byrd, D.R. and Matson, S.W. (1997) Nicking by transesteri¢cation:
the reaction catalysed by a relaxase. Mol. Microbiol. 25, 1011^1022.
[112] Marvin, D.A., Hale, R.D., Nave, C. and Helmer-Citterich, M.
(1994) Molecular models and structural comparisons of native and
mutant class I ¢lamentous bacteriophages Ff (fd, f1, M13), If1 and
IKe. J. Mol. Biol. 235, 260^286.
[113] Surette, M.G. and Stock, J.B. (1996) Role of alpha-helical coiledcoil interactions in receptor dimerization, signaling, and adaptation
during bacterial chemotaxis. J. Biol. Chem. 271, 17966^17973.
[114] Jelesarov, I., Durr, E., Thomas, R.M. and Bosshard, H.R. (1998)
Salt e¡ects on hydrophobic interaction and charge screening in the
folding of a negatively charged peptide to a coiled coil (leucine
zipper). Biochemistry 37, 7539^7550.
FEMSLE 11051 27-6-03