Download Two-step and one-step secretion mechanisms in Gram

Biochem. J. (2010) 425, 475–488 (Printed in Great Britain)
475
doi:10.1042/BJ20091518
REVIEW ARTICLE
Two-step and one-step secretion mechanisms in Gram-negative bacteria:
contrasting the type IV secretion system and the chaperone-usher pathway
of pilus biogenesis
Ana Toste RÊGO1 , Vidya CHANDRAN1 and Gabriel WAKSMAN1,2
Institute of Structural and Molecular Biology, Birkbeck and University College London, Malet Street, London, WC1E 7HX, U.K.
Gram-negative bacteria have evolved diverse secretion
systems/machineries to translocate substrates across the cell
envelope. These various machineries fulfil a wide variety of
functions but are also essential for pathogenic bacteria to infect
human or plant cells. Secretion systems, of which there are
seven, utilize one of two secretion mechanisms: (i) the onestep mechanism, whereby substrates are translocated directly
from the bacterial cytoplasm to the extracellular medium or
into the eukaryotic target cell; (ii) the two-step mechanism,
whereby substrates are first translocated across the bacterial inner
membrane; once in the periplasm, substrates are targeted to one
of the secretion systems that mediate transport across the outer
membrane and released outside the bacterial cell. The present
review provides an example for each of these two classes of
secretion systems and contrasts the various solutions evolved to
secrete substrates.
INTRODUCTION
Two main mechanisms for transport operate in the various
secretion systems described above: one-step or Sec-independent
compared with two-step or Sec-dependent transport mechanisms.
The T2SS, T5SS and the CU systems lack an inner membrane
transporter that can transfer substrates across the inner membrane
in response to ATP. For the first step of transfer across the inner
membrane, these systems require either the general secretory
pathway, also called Sec translocon, or the Tat pathway in the case
of some T2SS substrates. Specific secretion system machineries
are then used for transport of substrates from the periplasm across
the outer membrane. These systems thus utilize a two-step or
Sec-dependent mechanism for translocation. On the other hand,
T1SS, T3SS, T4SS and T6SS all utilize a simultaneous onestep translocation of substrates across both membranes. These
are thus Sec-independent secretion pathways. The CU pathway
is a very simple two-step translocation pathway requiring just
two specialized proteins: the periplasmic chaperone and an outer
membrane usher [10,11]. The T4SS, on the other hand, is
a complex assembly of 12 or more proteins spanning across
both the membranes with cytoplasmic ATPases and inner and
outer membrane proteins assisting substrate secretion and pilus
biogenesis [12]. The CU pathway and T4SS are thus examples
of two types of secretion nanomachines, one utilizing a two-step
mechanism for secretion (the CU pathway) and the other a onestep one (T4SS). In the present review, we use these two systems to
compare and contrast the architectures and secretion mechanisms
of these two classes of secretion systems. Other secretion systems
could have been chosen for the same purpose, but describing them
all would have made for a large and unwieldy review, the length of
which was beyond the limits imposed for reviews published in this
journal. Instead, the authors have opted for a review comparing
Gram-negative bacteria have evolved several simple to complex
machineries for transport of substrates across their cell membrane
in response to various environmental cues. These machineries,
known as secretion systems, are essential for bacterial virulence
and are used by bacteria to acquire nutrients, transport various
proteins, nucleic acids or toxins or assemble cell-surface
organelles such as pili or fimbriae. Transport has to occur across
both the membranes with the periplasmic space enclosed within.
To achieve this, bacteria have evolved at least seven different
secretion systems classified as type I (T1SS) to type VI (T6SS)
secretion systems and the CU (chaperone-usher) system [1,2].
T1SS is a tripartite system made up of an inner membrane ABC
(ATP-binding cassette) transporter, an adaptor protein bridging
the two membrane components and an outer membrane pore [3].
The T2SS (type II secretion system) spans both the membranes but
require the Sec or Tat pathway for transporting substrates across
the inner membrane [4]. The T3SS (type III secretion system)
or injectisome forms large supramolecular structures spanning
both membranes and is structurally and evolutionarily related
to bacterial flagella [5]. The T4SS (type IV secretion system)
also forms large assemblies spanning both membranes and is
evolutionarily related to the conjugation machinery [6,7]. The
T5SS (type V secretion systems) are autotransporters or twopartner secretion systems assembled only in the outer membrane
[8]. The T6SS is a recently discovered multi-protein system
still largely uncharacterized [2]. Finally, the CU system is a
simple system assembled in the outer membrane and responsible
for assembly and secretion of virulence surface organelles
[9,10].
Key words: chaperone-usher, protein secretion, structural biology,
type IV secretion, virulence factor.
Abbreviations used: AAD, all-α-helical domain; CU, chaperone-usher; DSC, donor-strand complementation; DSE, donor-strand exchange; EM, electron
microscopy; I-layer, inner layer; NBD, nucleotide-binding domain; Nte, N-terminal extension; O-layer, outer layer; T1SS, T2SS etc., type I secretion system,
type II secretion system etc; TM, transmembrane.
1
These authors contributed equally to the present review.
2
To whom correspondence should be addressed (email [email protected]).
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A.T. Rêgo, V. Chandran and G. Waksman
the one- and two-step secretion systems they study, i.e. the CU
pathway and the T4SS.
CU SECRETION SYSTEM
The CU system is responsible for the assembly and secretion
of pili (also called fimbria), multisubunit cell surface appendages
present in a wide range of pathogenic Gram-negative bacteria (e.g.
Escherichia coli, Salmonella enterica, Yersinia species, Klebsiella
pneumoniae, Pseudomonas aeruginosa etc.) [13]. CU pili are
virulence factors employed by bacteria for host recognition and
attachment, invasion and biofilm formation.
CU pili are polymers of protein subunits. Two specialized
proteins are required for CU pilus assembly: a periplasmic
chaperone that stabilizes pilus subunits and primes them for
subsequent polymerization into a pilus [14], and an outer
membrane usher that works as the assembly platform where
the chaperone subunit complexes are recruited, the subunits are
polymerized and translocated for presentation at the cell surface
[10,11].
CU pilus morphology varies from linear thick rods to more
flexible thin structures. Phylogenetic analysis of usher sequences
classified them into six major clades: α, β, γ (subdivided into γ 1 ,
γ 2 , γ 3 and γ 4 ), κ, π and σ fimbriae [15]. Rod-like fimbria, also
called ‘typical’ fimbria (found in α, γ and π clades) are one of the
best studied morphology types of the CU family, mainly through
extensive studies of P and Type 1 pili (π and γ 1 fimbrial clades
respectively) in UPEC (uropathogenic E. coli).
Architecture of P and Type 1 CU pili
CU fimbriae are typically encoded in individual gene clusters,
where all subunits in addition to the chaperone and usher are
encoded. The number of subunits varies from system to system.
For instance, the Type 1 pili cluster codes for four known subunits
(Fim A, F, G and H), whereas the P pili cluster codes for six known
subunits (Pap A, H, K, E, F and G). In both systems, these subunits
are arranged into two sub-assemblies: the pilus ‘rod’ and the ‘tip
fibrillum’ (Figure 1). The rod is a long thick rigid structure with
a ∼ 6.8 nm diameter made up of repeating subunits of PapA or
FimA (>1000 copies), in P and Type 1 pili respectively. These
repeating subunits form a right-handed helical structure with 3.3
subunits per turn [16,17]. The tip fibrillum of the P pilus is a thin
and flexible structure with a ∼ 2 nm diameter, made of repeating
PapE subunits (5–10 subunits) arranged in an open helical form
and connected to the rod by a copy of the adaptor subunit PapK
(Figure 1). A second single copy adaptor subunit PapF bridges
the distal end specialized adhesin subunit PapG with the PapE
polymer [18]. PapG has been shown to specifically recognize
the globo-series of glycolipids present in the human kidney, and
PapG has been shown to be a major virulence factor in kidney
infections [19,20]. In Type 1 pili, the linear tip fibrillum is simply
formed by one copy of each of the subunits FimF, FimG and
the adhesin FimH [21,22] (Figure 1B). For that reason, Type 1
pili form shorter tip fibres. FimH recognizes mannose residues
present in the human bladder [23] and Type 1 pili have been
shown to be responsible for bladder infections (cystisis), as well
as being implicated in attachment to abiotic surfaces in a model
biofilm system [24,25]. The P pilus rod is terminated at the base
by the PapH subunit that anchors the pilus structure to the cell
wall [26,27] (Figure 1A). No similar subunit has yet been found
in the Type 1 pili.
c The Authors Journal compilation c 2010 Biochemical Society
Figure 1
General diagram of the CU mechanism
(A) Diagram of the P pilus representing all the subunits involved at the outer membrane. Each
subunit is represented by oval shapes with a distinct colour and a letter; for example PapG
is labelled G, Pap F is labelled F, and so forth. (B) Diagram of Type 1 pili with the same
colour scheme as in (A). The pilus biogenesis is initiated by the export of the unfolded pilus
subunits to the periplasm via the Sec translocation machinery, schematically represented in the
inner membrane [155]. The periplasmic chaperone then captures subunits at the exit of the Sec
translocon and assists in their folding and targeting to the outer membrane dimeric usher where
one of the pores is activated. Once at the activated pore, subunits polymerize in an ordered
sequence until pili completion. The extracellular, outer membrane, periplasmic space, inner
membrane and cytoplasm are labelled E, OM, P, IM and C respectively.
Assembly of CU pilus
The initial step of the pilus biogenesis is the export of the pilus
subunits to the periplasm via the Sec translocation machinery
(Figure 1) (Sec-mediated transport through the inner membrane
is not within the scope of the present review. For excellent reviews
on this aspect, we refer the reader to [28,29]). At the exit of the Sec
machinery, each subunit interacts with a periplasmic chaperone
(PapD and FimC for P and Type 1 pilus subunits respectively)
for proper folding and guidance to the outer membrane usher.
In the absence of the chaperone, subunits aggregate and DegP
protease transcription is activated, promoting subunit degradation
[14,30,31].
Two-step versus one-step secretion systems
Figure 2
477
DSC and DSE
(A) DSC. Left panel: topology diagram of PapK (yellow) showing the PapD G1 (red) in the gap between strand A1 and F. Middle panel: structure of the PapD–PapK complex (PDB: 1PDK). PapD and
PapK are in ribbon and surface representations respectively, colour-coded red and yellow respectively. The location of the P1 to P4 pockets in the subunit’s groove as well as that of the conserved
chaperone residues co-ordinating the subunit’s C-terminus (Arg8 and Lys112 ) area are indicated. Right panel: close-up view of the P5 pocket of PapK. (B) DSE. Left panel: topology diagram of PapF
(in green) bound to PapD (in red). Middle panel: topology diagram of the same subunit (in green) bound to Nte of PapK (in yellow). Right panel: surface representation of PapF bound to the Nte of
PapK (PDB: 1N12). The location of the P2 to P5 pockets is shown. (C) The PapD–PapH structure (PDB: 2J2Z). Left panel: PapD and PapH are in surface and ribbon representations respectively,
colour-coded in red and grey respectively. The location of the P2 to P5 pockets is shown. Right panel: a close-up view illustrating the fact that PapH does not have a P5 pocket.
Mechanism of chaperone function: donor strand complementation
CU chaperones have a conserved boomerang-like structure,
composed of two Ig-like domains [32,33]. They possess two
conserved residues essential for forming complexes with the
subunits (Figure 2A). These residues are located between the two
domains of the chaperone. In the PapD chaperone, these residues
are Arg8 and Lys112 , and other chaperones have homologous
residues in the same position [33,34]. Mutations in these
residues abolishes pilus biogenesis [33]. Formation of chaperone–
subunit complexes is also dependent upon a series of alternating
hydrophobic surface residues (P1 to P4 residues) located in the
last β-strand of the chaperone’s N-terminal domain (strand G1 )
(Figure 2A).
Subunits share an incomplete Ig-like structure [35,36] with
only six of the seven characteristic β-strands (βA to βF) present
in the fold (Figures 2A and 2B). The C-terminal β-strand (βG)
is entirely missing. As a result, a hydrophobic groove in the
structure is created where the missing secondary structure should
be. Because of the missing strand, subunits are unstable and
unable to fold correctly on their own. Folding and stabilization
of subunits is afforded by the insertion of the chaperone’s G1
strand into the subunit’s hydrophobic groove and interaction of
the conserved arginine and lysine residues with the C-terminal
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A.T. Rêgo, V. Chandran and G. Waksman
carboxylic group of the subunit’s strand F (Figure 2A). Insertion
of the G1 strand results in its P1 to P4 residues occupying
the P1 to P4 sites/pockets of the subunit’s groove (Figure 2A).
Thus the chaperone ‘donates’ in trans the missing secondary
structure: this mechanism of chaperone-mediated subunit folding
and stabilization is termed DSC (donor-strand complementation)
[14,30,35–37]. The resulting complemented fold, however, is not
a canonical Ig-fold, since the chaperone G1 strand runs parallel to
the subunits F strand instead of anti-parallel [38].
Mechanism of subunit polymerization: donor-strand exchange
Every pilus subunit possesses a 10–20 residue long disordered
Nte (N-terminal extension), which, similar to the G1 β-strand of
the chaperone, contains alternating hydrophobic residues. This
Nte was shown not to be part of the Ig-like fold [36]. During
the course of polymerization, the Nte of the incoming subunit
displaces the G1 donor β-strand of the chaperone, complementing
the Ig-like fold of the previous subunit assembled in the pili
[39] (Figure 2B). This mechanism of subunit polymerization
is called ‘donor-strand exchange’ (DSE) [35,36,39,40]. In the
subunit groove, the chaperone G1 strand hydrophobic residues
occupy the P1 to P4 sites/pockets, as mentioned above, whereas
after DSE the incoming Nte hydrophobic residues (termed P2
to P5 residues) occupy sites/pockets P2 to P5 of the groove
(Figures 2A and 2B). The subunit fold is now a canonical Igfold, since the Nte strand runs anti-parallel to the subunit’s F
strand, thus in a more energetically favourable conformation.
The reaction by which the chaperone G1 strand is substituted
by the incoming subunit Nte in the groove proceeds via a zipin-zip-out mechanism, whereby the process of DSE is initiated
by the insertion of the P5 residue of the incoming subunit’s Nte
into the P5 pocket of the previously assembled subunit groove
[41,42] (see Figure 2A, right panel for the P5 pocket). DSE
progresses with the new Nte gradually displacing the chaperone’s
G1 strand step by step from P5 to P1 [42] (Figure 2B). The
receiving subunit structure then transitions from a chaperonestabilized semi-unfolded state of high-energy to a folded state of
lower energy when in interaction with an other subunit [43]: it
is believed that it is the folding energy released upon DSE that
drives subunit polymerization [43,44]. No other external source
of energy is required to drive pilus polymerization. Termination
of pilus biogenesis happens upon incorporation of the subunit
PapH in the growing pilus. This happens because PapH is unable
to undergo DSE since it lacks a P5 pocket [27] (Figure 2C). No
mechanism for type 1 pilus termination has been found yet as a
PapH-like termination subunit is unknown in that pilus system.
The usher assembly platform
In vivo DSE occurs at the outer membrane usher [44]. DSE is
observed in vitro and in the absence of the usher, but proceeds
only at very slow rates [45]. Rapid fibre formation, even in vitro,
requires the presence of the usher. Thus the usher acts as a catalyst
of DSE [46].
Outer membrane ushers are ∼ 90 kDa proteins with four distinct
functional domains: an N-terminal periplasmic domain with
∼ 125 residues, a large translocation pore domain with ∼ 500
residues that is interrupted by a soluble plug domain of ∼ 110
residues and a C-terminal periplasmic domain with ∼ 170 residues
[47,48] (Figure 3A).
The N-terminal domain binds chaperone–subunit complexes
[49,50] with binding affinities for each of the complexes
decreasing in an order reflecting the natural order of subunits
c The Authors Journal compilation c 2010 Biochemical Society
in the pilus, a process that is believed to be responsible, in
part, for subunit ordering. Structural and biophysical studies
of the N-terminal domain of the FimD usher bound to both
FimC–FimH and FimC–FimF showed that a flexible tail at the
N-terminus (residues 1–24) was responsible for 40%–50 % of
the binding interface and that tail was directly interacting with
the subunit (FimH or FimF) (Figure 3B). Deletion mutants of
residues 1–24 abolished chaperone–subunit interaction with the
usher [49,51]. Similarly, for the PapC N-terminal domain, deletion
of residues 1–11 abolished targeting of the usher to chaperone–
subunit complexes [50]. Thus the tail of the N-terminal domain
seems to be responsible for subunit-specific interactions. In vitro,
the N-terminal domain doesn’t seem to be fundamental for pilus
biogenesis, which may suggest that its role in vivo may be to
capture chaperone–subunit complexes and increase their local
concentration to accelerate pilus assembly [53].
The usher translocation pore forms an outer membrane
β-barrel containing 24 TM (transmembrane) β-strands [52,53]
(Figure 3C). The pore has an inner diameter of 45 Å×25 Å
(1 Å=0.1 nm), a size compatible with the passage of folded
subunits. Between strands 6 and 7 lays the plug domain, a βsandwich fold that, in an inactive usher, completely occludes the
pore, preventing the passage of the subunits. Deletion of this
domain completely abolishes the assembly of pili in vitro [53],
thus the plug domain participates actively in pilus biogenesis.
The C-terminal domain role is still not completely understood,
but C-terminal truncation and deletion mutants of the P pilus
usher PapC demonstrated that the C-terminus is fundamental
for proper binding of chaperone–subunit complexes and pilus
assembly [53,54]. Differences in protease susceptibility in the
Type 1 pilus usher FimD show that following targeting to the usher
N-terminus, the N-terminal domain of FimH (from the
FimC–FimH complex) forms stable interactions with the FimD
C-terminus, inducing a conformational change in FimD that may
be fundamental in the activation step of pilus biogenesis [55–
57]. Such conformational change must include the movement of
the plug either inwards within the translocation pore or outwards
in the periplasm [52]. Binding of the chaperone-adhesin (PapDPapG) with the usher C-terminus was also demonstrated in vitro.
Complementation studies on PapC C-terminal truncates have
also shown that, in the absence of the C-terminal domain, the
translocation pore is unable to become activated. These results
suggest an important role of the C-terminal domain in the first
step of pilus biogenesis, the activation of the usher [54].
The P pilus usher PapC assembles into ring-shaped homodimeric functional units in vivo. This was demonstrated
by cryo-EM (where EM is electron microscopy) images of
two-dimensional crystals where PapC was reconstituted in
E. coli lipids and in complementation studies of PapC lossof-function mutants [54,58]. Dimer formation doesn’t seem
to be necessary for fibre formation in vitro [53]. Cryo-EM
reconstruction of FimD–FimH–FimG–FimF–FimC shows
that only one of the ushers is activated and able to secrete
subunits (Figure 3D) [52]. In fact, the requirement for a
second usher in vivo is not completely understood. Remaut
et al. [52] proposed a model where the two usher N-terminal
domains alternate in the recruitment of new chaperone–subunit
complexes. The structure of the C-terminal domain and of
usher–chaperone–subunit complexes will provide answers to the
mechanism of usher activation and assembly mechanism.
TYPE IV SECRETION SYSTEM
T4SSs are specialized assemblies utilized by both Grampositive and Gram-negative bacteria for one-step transport of
Two-step versus one-step secretion systems
Figure 3
479
The usher PapC structure
(A) Domain organization of PapC. The translocation domain and the N-terminal domains (Ntd) are represented in dark blue, the plug domain in magenta, and the C-terminal domain (Ctd) in white.
Residue boundaries are indicated. (B) Structure of the FimD N-terminal domain (dark blue) bound to the FimC–FimHp (red and light green respectively) complex (PDB: 1ZE3). FimHp is the pilin
domain of FimH. (C) Structure of the translocation domain of the PapC usher (PDB: 2VQI). The domain is in ribbon representation colour-coded in dark blue for the barrel, yellow for the single helix
present in the structure, orange for the trigger hairpin, and magenta for the plug domain. Upper panel: side views. Lower panels: top views. (D) Type 1 pili tip captured by cryo-EM. X-ray structures
from FimH (light green), FimD (dark blue), FimG (orange), FimC (red) and FimF (yellow) were docked [52]. X-ray crystallography structures used were FimH (PDB: 1QUN), FimD (PDB: 2VQI),
FimG (PDB: 3BFQ), FimC–FimF–FimDN (PDB: 3BWU) and the cryo-EM map of the FimD2:C:F:G:H type 1 pilus tip assembly intermediate is from emd_5009 (EM Data Bank). A three-dimensional
interactive Figure of this structure is available at http://www.BiochemJ.org/bj/425/0475/bj4250475add.htm.
protein, nucleic acid or nucleoprotein substrates across cell
membranes [6,7,59,60]. They are evolutionarily related to
bacterial conjugation systems. T4SSs are implicated in the
transport of virulent proteins or DNAs in various plant, animal or
human pathogens [61,62], in bacterial conjugation and in DNA
uptake or release into the extracellular space [63,64]. T4SSs
facilitate the exchange of genetic material, promoting genetic
plasticity and adaptive response of bacteria to the environment.
Conjugative transfer of antibiotic-resistance genes via T4SSs is a
leading cause for emergence of multidrug resistance [65].
T4SSs are formed by at least 12 proteins, termed VirB1–
VirB11 and VirD4 according to the prototypical T4SS from
Agrobacterium tumefaciens, although some T4SSs are composed
of just a subset of these proteins [60,66]. The VirB/VirD4
proteins can be grouped based on their function and location
into energizing proteins, core translocation pore complex, pilusassociated proteins and other T4SS proteins. Energizing proteins
include the ATPases, VirB4, VirB11 and VirD4. VirB6 to VirB10
proteins form the core translocation complex. VirB6, VirB8
and VirB10 are inner membrane proteins, whereas lipoproteins
VirB7 and VirB9 were thought to form the outer membrane
pore. Most T4SSs form an extracellular pilus formed by the
major VirB2 and minor VirB5 proteins. The other T4SS proteins
include the putative lytic transglycosylase VirB1 and membrane
protein VirB3, the location and function of which are still either
ambiguous (location) or unknown (function).
Structure and role of the various subunits
The VirB4, VirB11 and VirD4 ATPases
The three ATPases VirD4, VirB4 and VirB11 power T4SSs. They
are located in the cytoplasm, sometimes inserted in the inner
membrane, and form a network of interactions among themselves
and the rest of the assembly [67].
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480
Figure 4
A.T. Rêgo, V. Chandran and G. Waksman
Atomic structures of known T4SS components
Top panels, the crystal structures in cartoon representation for the E. coli VirB5 homologue TraC (PDB: 1R8I), B. suis VirB8 (PDB: 2BHM), H. pylori VirB10 homologue ComB10 (PDB: 2BHV) and the
NMR structure of E. coli VirB9/TraO C-terminal domain in complex with VirB7/TraN (PDB: 2OFQ). The lower panels show in cartoon representation, the oligomeric and monomeric crystal structures
of the H. pylori VirB11 homologue HP0525 (PDB: 1NLZ) and the E. coli VirD4 homologue TrwB from plasmid R388 (PDB: 1E9S). CTD, C-terminal domain; NTD, N-terminal domain.
VirD4
VirD4 proteins are the cytoplasmic gatekeepers that recruit T4SS
substrates to the T4SS channel. VirD4s are inner membrane
proteins with an N-terminal TM domain and a large cytoplasmic
domain [68]. TrwB, encoded by the IncW conjugative plasmid
R388, has been the most characterized VirD4 homologue. The
TM domain of TrwB plays a role in stability, substrate selectivity
and oligomerization of the protein [69,70]. The crystal structure
of the cytoplasmic soluble domain of TrwB [71] revealed a large
110 Å × 90 Å globular hexameric assembly with a 20 Å channel
(Figure 4). This channel narrows down to an ∼ 8 Å constriction
at the cytoplasmic end. Each subunit is shaped like an orange
slice and made up of two distinct domains, an NBD (nucleotidebinding domain) and an AAD (all-α-helical domain). The NBD
domain is a mixed α/β RecA-like domain with a central highly
twisted nine-stranded β-sheet flanked by α-helices on both sides
and orientated towards the inner membrane. Both the Walker A
and B motifs for NTP-binding are essential for secretion [72]. A
DNA-dependent ATPase activity has been demonstrated for TrwB
[73]. No helicase activity has been observed so far for VirD4s.
The AAD domain, consisting of seven α-helices, is orientated
towards the cytoplasm. A DNA-binding role for this domain has
been suggested, based on structural homology.
VirB11
VirB11 belongs to a large family of traffic ATPases involved
in various secretion systems such as type II, III, IV and VI
[66,74,75]. These NTPases are important for their ATP-bindingdependent substrate selection and translocation, and for pilus
production [76]. VirB11-like proteins exhibit distinct substrate
c The Authors Journal compilation c 2010 Biochemical Society
specificity and their NTPase activity is enhanced by the presence
of detergent/phospholipids in vitro [77,78]. VirB11-like proteins
have indeed been shown to be peripheral membrane proteins associated with the inner membrane [79]. Crystal structures have been
solved for the Helicobacter pylori VirB11 homologue HP0525
in its apo and substrate-bound forms, and for the Brucella suis
VirB11 protein [80–82] (Figure 4). Each HP0525 monomer is
made up of two domains: an N-terminal domain with a novel fold
and a C-terminal domain with the typical RecA fold. The two
domains form hexameric rings mounted on each other shaped like
a six-clawed grapple. The N-terminal domain consists of a central
six-stranded β-sheet packed against two α-helices on one side,
whereas the C-terminal domain is made up of a seven-stranded
β-sheet sandwiched between three and four α-helices on either
side. The interface between the two domains forms the NTPbinding site. VirB11 from B. suis shows a very similar double
hexameric ring structure with the individual domains in each
monomer structurally similar to those of HP0525 [82]. However,
a large domain swap is noticed that leaves the overall hexamer
intact but alters both the substrate-binding site and inter-subunit
interfaces.
The apo and substrate-bound structures of HP0525 have
given valuable structural insight into the function of VirB11like ATPases [80,81,83]. The apo-form of HP0525 shows an
asymmetric hexamer due to rigid body movements in the NTD
ring of the assembly. The conformational flexibility has been
suggested to help further interactions with the rest of the T4SS
assembly. ATP binding takes place initially at three catalytic sites,
giving rise to a more rigid conformation to the entire assembly.
ATP hydrolysis at these sites followed by ATP binding to the
remaining three sites locks the entire assembly into a perfectly
Two-step versus one-step secretion systems
symmetrical hexamer. Further hydrolysis and release of ADP from
all sites returns the molecule to its initial asymmetric apo-form.
VirB4
VirB4-like proteins are the largest ATPases in T4SSs and are
evolutionarily conserved in both Gram-negative and Grampositive bacteria [6,84]. Bioinformatic studies suggest four conserved motifs, including the Walker A and B motifs and a
conserved domain for the VirB4 family of proteins [85]. The NTPbinding motifs are essential for the functionality of T4SSs [86].
However, unambiguous demonstration of the ATPase activity for
VirB4 homologues has been difficult, with two positive reports
for A. tumefaciens VirB4 and more recently TrwK from plasmid
R388 [87,88].
No structural detail is available for VirB4-like proteins and
there is ambiguity regarding the exact cellular location and oligomerization state. Topological studies on A. tumefaciens VirB4 map
it as a polytopic cytoplasmic membrane protein with probably two
periplasmic and three cytoplasmic domains [89]. TrwK, the VirB4
homologue from plasmid R388, however, is a soluble protein [88].
A hexameric model of the C-terminal domain of VirB4 has been
proposed based on its relationship to the cytoplasmic domain
of VirD4 homologue TrwB by divergent evolution [90]. This
initial model suggested a cytoplasmic location for VirB4, although
subsequent work places the C-terminal domain predominantly in
the periplasm [91].
Core channel components
VirB6
VirB6 is a highly hydrophobic inner membrane channel subunit
essential for T4SS. No structural information is available to
date for this T4SS component. Topological studies on both
A. tumefaciens VirB6 and H. pylori ComB6 have mapped an
N-terminal periplasmic domain, five TM segments and a cytoplasmic C-terminal domain [92,93]. A large periplasmic loop (P2
loop) in the centre of the protein between TM2 and TM3 presumably contributes to the inner walls of the transport channel. The
C-terminal TM segments orchestrate substrate transfer to the other
inner membrane component VirB8, whereas both terminal regions
co-ordinate with the outer membrane and pilin subunit VirB9 and
VirB2. The physiological oligomeric state of VirB6 itself is not
known.
VirB8
VirB8 is a bitopic inner membrane protein with a short
cytoplasmic tail, a single TM helix and a large periplasmic
domain [68]. VirB8 is thought to serve as the nucleation centre
for the assembly of T4SSs [60,94]. Immunofluorescence and
immunoelectron microscopic methods showed unique clustering
of VirB8 gold particles independent of other VirB proteins
and also the influence of VirB8 on VirB9–VirB10 localization
[94]. VirB8 participates in a rich network of protein–protein
interactions in the T4SS with VirB1, VirB4, VirB5, VirB9, VirB10
and VirB11, and is in direct contact with the substrate during
transport [94–100].
High-resolution structural information is available for the
periplasmic domains of two homologues, A. tumefaciens and
B. suis VirB8 (Figure 4) [101,102]. Both crystal structures reveal
a dimer of VirB8 with an extended four-stranded antiparallel
β-sheet juxtaposed by five α-helices. The periplasmic domains
have a fold similar to nuclear transport factor NTF2. The crystal
481
structures reveal a deep groove lined by highly conserved residues,
a structural feature absent in NTF2 and other similar fold proteins
and is probably a hot spot for protein–protein interactions.
Mutational analysis of VirB8 identified a β-sheet region adjacent
to this groove to be important for interactions with VirB4 and
VirB10 [103]. The physiological oligomeric state of this protein
in the T4SS assembly is still unknown.
VirB10
VirB10 is also a bitopic inner membrane protein with a similar
topology as VirB8 with a short N-terminal cytoplasmic tail,
a single predicted TM helix and a large periplasmic domain
consisting of a proline-rich region and a β-barrel domain [68,104].
VirB10, although inserted in the inner membrane, has also been
observed associated with the outer membrane [94]. Interactions
of VirB10 with VirB9 [100] and the energizing proteins VirB4,
VirB11 and VirD4 [67,105–108] substantiated by mutational
analysis have established VirB10 as the structural scaffold of
T4SSs bridging the two membranes together [104].
A high-resolution crystal structure is available for part of the
periplasmic domain of H. pylori ComB10 protein [102] (Figure 4).
This C-terminal periplasmic domain consists of an atypical
β-barrel flanked by an α-helix on one side and an α-helical
antennae-like projection at the top. The altered β-barrel is splayed
open on one side. A head to tail dimer is observed in the crystal
structure, which is probably a crystal-packing artefact. Mutational
studies have highlighted the role of the side helix and α-helical
antennae in uncoupling pilus biogenesis and substrate transfer
functions of T4SSs [104]. See Note added in proof.
VirB10 shares features with the inner membrane energy sensor
protein TonB: both are bitopic membrane proteins with a prolinerich region and conserved hydrophobic or coiled-coil regions
important for protein–protein interaction and function. Similar to
TonB, VirB10 probably couples inner membrane energy status
to the outer membrane gating required for transport. Indeed,
VirB10 responds to the cellular ATP levels with different protease
susceptibility, suggesting different structural conformations. The
structural transition of VirB10 in response to ATP levels also
affects the VirB7–VirB9 heterodimer and VirB7–VirB9–VirB10
complex formation [109].
VirB7 and VirB9
VirB7 is a monotopic lipoprotein [110]. The modified N-terminus
is anchored in the outer membrane and the C-terminus is
periplasmic. In A. tumefaciens, VirB7 stabilizes VirB9 by forming
a disulfide link between the two proteins [111]. VirB9 is mainly
a periplasmic protein that has been suggested to form the outer
membrane pore, although no TM segments have been predicted.
Mutational studies on A. tumefaciens VirB9 has mapped the
N-terminal domain to be important for substrate selection and
the C-terminal domain for stabilizing the VirB10–VirB9–VirB2
proteins that form the distal end of the channel and are essential
for secretion [112]. The NMR structure has been solved for
the interacting domains, for example the C-terminal domain
of the VirB9 homologue encoded by the E. coli plasmid pKM101,
TraO (TraO-CT), bound to the VirB7 homologue TraN encoded
by the same plasmid (Figure 4) [113]. The structure reveals a
β-sandwich fold for TraO-CT, around which TraN wraps. Two
edge strands in TraO that diverge away from the β-sandwich
fold form an appendage which has been shown to be surface
exposed in A. tumefaciens, suggesting a role in outer membrane
pore formation. See Note added in proof.
c The Authors Journal compilation c 2010 Biochemical Society
482
A.T. Rêgo, V. Chandran and G. Waksman
Pilus components
The function of the type IV secretion pilus (not to be mistaken
for the type IV pilus, the assembly of which is a T2SSrelated process) is still unclear. It might be used as conduit
for substrates [13,114] or adhesion [115,116]. The T pilus of
A. tumefaciens consists of a processed form of VirB2, the major
component of pilin, and VirB5, the minor component [117,118].
Pilus biogenesis requires other T4SS components and several
uncoupling mutations have been mapped that allow secretion but
not pilus assembly [76,104,112]. VirB2 is highly processed: the
N-terminal leader signal peptide that targets the pilus subunit
across the inner membrane is cleaved by specific peptidases and
further processing might occur, sometimes including cyclization
[119–122]. No structural information for VirB2-like proteins
alone is available. However, a crystal structure is available for
the VirB5 homologue in pKM101, TraC (Figure 4C) [123].
The structure reveals a three-helix bundle capped by a globular
appendage. Structure-based mutagenesis studies suggested a role
of VirB5 in adhesion and host recognition [123]. Recently,
the F pili structure has been determined using cryo-EM and
single-particle reconstruction [124]. Two pilin subunits pack with
two different symmetries that co-exist; a C4 symmetry forming
stacked rings axially separated by ∼ 12.8 Å and a one-start helical
symmetry with an axial rise of 3.5 Å per subunit and a pitch of
∼ 12.2 Å. The central lumen seems large enough for transferring
single-stranded DNA but not folded proteins.
Other T4SS-associated proteins
VirB1
VirB1, although non-essential for secretion, is essential for
pilus biogenesis [125–127]. Deletions of the virB1 gene lead to
attenuated virulence with respect to T4SS functions [99]. An
exception in this case is that of H. pylori, where the VirB1
homologue, HP0523, is essential to bacterial virulence [128].
VirB1 is a periplasmic protein with a conserved lysozyme-like
transglycosylase domain [129]. The catalytic activity of the transglycosylase domain to lyse the peptidoglycan cell layer probably
helps to accommodate the large T4SS assembly in the periplasmic
space. VirB1 is processed [130] to release a C-terminal third of
the protein VirB1* which is required for pilus biogenesis and is
secreted to the extracellular space [127]. Two hybrid experiments
and later biochemical experiments have shown interaction of
VirB1 with other T4SS components VirB9, VirB8 and VirB11
suggesting more (as yet undefined) roles for VirB1 in T4SS
assembly and function [99].
VirB3
VirB3 is one of the most ill understood proteins in the T4SS
assembly both in terms of localization and function. It is
a membrane protein with TM segments predicted near the
N-terminus. Localization of this protein is very uncertain, with
reports suggesting either inner or outer membrane localization
[131,132]. Chimaeric VirB3/VirB4 proteins previously identified
in certain T4SS assemblies such as Campylobacter jejuni [133]
suggest an inner membrane localization to be more probable due
to association with VirB4, which is associated with the inner
membrane. However, no functional role has been identified so far
for this protein.
Assembly of T4SSs
The biogenesis of the T4SS assembly has been proposed to start
with the formation of a core complex (VirB6–VirB10) (Figure 5),
c The Authors Journal compilation c 2010 Biochemical Society
followed by recruitment of the ATPases (VirD4, VirB4 and
VirB11) and/or pilus biogenesis (VirB2 and VirB5). Secretion
and pilus biogenesis might require two different complexes [6,7],
and thus, after assembly of the core complex, there might be
a bifurcation in the assembly process leading to formation of
two different complexes, one used for secretion, the other used
for pilus biogenesis. Indeed, mutations in some VirB proteins
decouple secretion from pilus biogenesis [76,92,104,112]. Also,
VirD4 is only used in secretion, whereas VirB11 is only used
in pilus biogenesis [127,134]. However, at this stage, the exact
composition of these complexes is not known.
A recent breakthrough in the field has been the 15 Å cryoEM structure of the core translocation channel formed by the
VirB7, VirB9 and VirB10 homologues of the E. coli pKM101
plasmid (TraN–TraO–TraF) (Figure 5) [135]. This provided the
first snapshot of a T4SS assembly. The core complex is a 1.05 MDa
structure with 14-fold symmetry and traverses across both inner
and outer membranes. The nucleation factor VirB8/TraE, although
co-expressed with the other proteins, is not required for forming
the core complex. The lipidation of lipoprotein VirB7/TraN is
also not essential for core complex formation, but required for
targeting the complex to the outer membrane.
The structure shows multilayered rings consisting of 14 copies
each of TraN/VirB7, TraO/VirB9 and TraF/VirB10, with a height
and diameter of 185 Å. Two distinct layers are seen, termed the I
(inner)- and O (outer)-layers with respect to location in the membrane (Figure 5). The two layers are partly or fully double walled.
The O-layer consists of a cap and a main body. The cap, which
probably traverses the outer membrane, encloses a 20 Å pore that
narrows to 10 Å at the junction with the main body. The main
body has a larger internal chamber that is 110 Å wide narrowing
down to 55 Å at the base. The I-layer, that resembles a cup, has
a partial double wall that merges down at the base and encloses a
chamber similar to the O-layer. A total of 14 finger-like densities
extend below the I-layer. Using various biochemical studies, the
C-terminal domains of both VirB9 and VirB10 have been located
in the O-layer and the N-terminal domains of both proteins in the
I-layer. The finger-like protrusions probably are the TM segments
of TraF inserted in the inner membrane.
T4SS mechanism
One of the most valuable contributions to the T4SS field has
been the TrIP (transfer DNA immunoprecipitation) study on the
A. tumefaciens T4SS, delineating the substrate transfer route
[126]. In A. tumefaciens, the substrate is the T-DNA (transferred
DNA). It successively contacts VirD4, VirB11, VirB6, VirB8,
VirB2 and VirB9 during transfer. Other T4SS components, VirB3,
VirB4, VirB5, VirB7 and VirB10, assist the transfer indirectly,
modulating/regulating differentially each of the transfer steps. The
coupling protein VirD4 contacts the substrate first, independently
of ATP hydrolysis [67]. The substrate is then passed on to VirB11,
independently of any energy requirements. The lipoprotein VirB7
is important for this initial step of transfer, highlighting its
importance for mediating the formation of the stable core
assembly. The substrate then comes in to contact with the inner
membrane proteins VirB6 and VirB8. This early transfer depends
on the energetic components VirD4, VirB4 and VirB11, and ATP
hydrolysis is required at this stage. This seems to suggest that
VirB6 and VirB8 function together and probably line the inner
surface of the VirB7–VirB9–VirB10 core complex to contact the
substrate directly. The substrate is then transferred to the outermembrane-associated proteins VirB9 and the major pilin subunit
VirB2. VirB3, VirB5 and VirB10 are important at this stage of
transfer. The importance of VirB10 is evident from the fact that it
Two-step versus one-step secretion systems
Figure 5
483
A model for Type IV secretion systems
A model for the assembly of T4SS components VirB1–VirB11/VirD4 is shown. A cut-away view of the cryo-EM structure of the core translocation assembly formed by VirB7/VirB9/VirB10 homologues
in E. coli pKM101 is shown in grey crossing both the inner and outer membranes. A surface representation of the atomic structures of T4SS components (VirB11 homologue, PDB entry 1NLZ; VirD4
homologue, PDB entry 1E9S; VirB5 homologue, 1R8I; and VirB8, PDB entry 2BHM) that are available are shown. The T4SS components for which no known structural information is available are
shown in distinct shapes and colours. The three ATPases powering the T4SS on the cytoplasmic side of the membrane are shown. Inner membrane components VirB6 and VirB8 are shown lining the
inner surface of the core complex. Pilus-associated proteins VirB2 and VirB5 and other proteins VirB1 and VirB3 are shown. All components are labelled according to their A. tumefaciens VirB/VirD4
nomenclature.
bridges the two membrane components together (Figure 5) [104]
and plays the role of an energy sensor for bringing about the
required conformational changes important for substrate transfer
[109]. VirB5 is a minor pilin subunit probably assisting the
passage of substrate via the pilus. Thus the entire pathway for
substrate translocation has been defined and, except for VirB1, all
proteins play an important role for secretion.
CONTRASTING THE TWO SYSTEMS
Simplicity compared with complexity in the translocation of
substrates
Secretion systems range from relatively simple structures, such as
the CU secretion system composed of two subunits (the chaperone
and the usher) specialized in assembling and driving the secretion
of pilus subunits, to more complex machineries such as T4SS,
which is composed of at least 12 subunits able to mediate secretion
of a wide variety of substrates. T4SS proteins span both the inner
and outer membranes, forming MDa multicomplex machineries,
whereas the CU system proteins only form complexes with the
chaperone when they are recruited, and then with the usher and
each other to form the pilus structure. The translocation pore
in the CU system is composed simply of a ∼ 180 kDa outer
membrane dimeric usher [52] that works as an assembly platform.
The pore diameter of one of the PapC monomers (45 Å × 25 Å)
is large enough to allow folded subunits translocation (ranging
from 27 Å × 22 Å to 32 Å × 23 Å in diameter for PapA and PapK
respectively) upon movement of the plug domain. How the plug
is let go is not known, but simple mechanical processes might
operate such a release, probably mediated by the interaction of
the first chaperone–subunit to be presented to the usher (the chaperone–adhesin complex) with the C-terminal domain of the
usher. In contrast, the translocation pore complex in T4SSs, in
its minimal form, involves at least three proteins (VirB7, VirB9
and VirB10) with 14 copies of each assembling into a 1.05 MDa
channel complex [135]. At least on one side (the O-layer), the
channel is closed and, thus, it must open to let substrates through.
This necessitates complex multi-proteins sensing and mechanical
devices to operate the conformational changes required to drive
secretion of substrates, probably mediated by VirB10, but also
other VirB proteins not contacting the substrate.
ATP dependency/independency
Protein secretion across the outer membrane is dependent on
an energy source. However, the most commonly used energy
store in living organisms, ATP, is not present in the bacterial
c The Authors Journal compilation c 2010 Biochemical Society
484
A.T. Rêgo, V. Chandran and G. Waksman
periplasm. In the T4SS several cytoplasmic components are
assembled in a large cytoplasmic/inner membrane ATPase
complex (VirD4, VirB4 and VirB11) that utilizes ATP to operate
the conformational changes required to drive secretion across
both the inner and outer membranes. In the CU system no
cytoplasmic or inner membrane components are encoded and no
ATP is used. In fact, no external source of energy is required for
DSE and consecutive pilus formation [44]. Instead, the chaperone
stabilizes a high-energy conformation of the bound subunit, which
upon DSE is released, resulting in an exceptionally stable and
closed subunit conformation. The free energy released during the
process is believed to be sufficient to drive pilus formation [43].
It should not be inferred from the comparison above that,
in all two-step secretion systems, transport through the outer
membrane is ATP-independent. For example, in type II secretion,
although substrates are dependent on Sec or Tat (and thus ATP) for
transport through the inner membrane, transport through the outer
membrane also requires ATP. Although this is not completely
understood, it has been proposed that passage through the outer
membrane pore (termed the ‘secretin’) is powered by pilus growth
and retraction, a process dependent on inner membrane ATPases
and consumption of ATP. Thus, in T2SS, an active ATP-dependent
mechanism utilizing the pilus as a piston might mechanically push
substrates through the outer membrane secretin [4]. Conversely,
not all one-step secretion systems are dependent on ATP. For
example, although one-step transport by some T1SS are powered
by ATP, other T1SSs might use the inner membrane proton
gradient as a source of energy [136,137]. The present review does
not have the space to expand the description of these systems, but it
must be realized that, although common features are clearly shared
among secretion systems, each also exhibit unique characteristics
[138].
Chaperone presence and dependency
Molecular chaperones have been characterized in all living
cells as accessory proteins in various cellular functions. These
include de novo protein folding, stabilization of proteins under
stress conditions and maintenance of a loosely folded state for
translocation across membranes. Cytoplasmic chaperones are
involved in different types of protein secretion. They have been
best characterized in T3SSs where they are thought to maintain
the stability and secretion competence of effector proteins, but
have also been found in the T4SS. For example, the VirE1 protein
of A. tumefaciens is necessary for the translocation of the effector
protein VirE2 into plant cells [139–143]. Other ‘type IV adaptor
proteins’ required for translocation of a subset of substrates, such
as IcmS, IcmW and LvgA proteins of the L. pneumophila Icm/Dot
T4SS [144–146] may have a similar function. CagF is another
candidate for secretion chaperone-like molecule (present in both
soluble and membrane-associated milieu) of the CagA protein
[147].
In the periplasm, chaperone function is distinct from the
cytoplasmic chaperones and independent of any known source
of cellular energy. PapD-like periplasmic chaperones are small
monomeric proteins that transiently contribute steric information
to pilus subunits to facilitate their folding, maintain them in a
high-energy state, and prime them for future assembly. In the CU
secretion system, the role and mechanism of the chaperone is
very well understood and a single chaperone seems to be enough
to drive all of the different subunits to the assembly platform.
On the other hand, in T4SSs and other systems, chaperones are
still not very well characterized and their function is still poorly
understood.
c The Authors Journal compilation c 2010 Biochemical Society
Type IV secretion pilus compared with CU pilus
T4SS pili are varied in shape and size. The conjugation pili
in T4SS systems are classified based on morphology into two
classes: the long flexible F-type pili and the short rigid P-type
pili [148]. The F-type T4SS pili measure 2–20 μm in length
and 8–9 nm in diameter with a 2 nm central lumen [149,150].
The P-type pili are shorter than 1 μm in length and 8–12 nm
in diameter [149,150]. The overall structure of a F-type pilus
has been determined (see above) and the subunits appear to
stack in a superhelical arrangement. On the other hand, the H.
pylori T4SS system encoded by the Cag pathogenicity island
produces distinctive, large sheathed pili, ∼ 100–200 nm long and
∼ 70 nm wide with a ∼ 45 nm central needle made up of a
VirB10 homologue (HP0527/CagY) [115,128,151]. It is believed
that T4SS pili essentially play an adhesion role. For example,
CagL, possibly the VirB5 homologue of the Cag-encoded T4SS,
is targeted to the pilus surface and appears to play a role in host
recognition [115]. However, the role of T4SS pili as adhesive
devices has not been as clearly demonstrated as in the CU pili
systems, notably the P and Type I CU pili systems. Thus it remains
unclear whether T4SS pili are used for adhesion or as conduit for
substrate secretion or both.
CU systems also encode a variety of pilus types ranging from
the rigid rod-like pili, the detailed description of which is provided
above (P and Type 1 CU pili), but also very thin fimbriae or
filaments forming mingled masses at the surface of the bacteria
that harbour them [40,42,152,153]. In P and Type 1 pili, only one
protein is responsible for adhesion and host recognition (PapG
and FimH respectively), and this protein locates at the pilus tip
due to the unique properties of the chaperone–adhesin complex
to activate the usher. However, in some other CU pili systems
where only one or two pilus subunits have been identified, these
are likely to combine the functions of structural and adhesive
subunits, i.e. they assemble to form a pilus or fimbria but also act
as adhesins. In that case, the adhesive property of the pilus would
no longer be confined to the tip but thought to be spread out over
the entire length of the pilus/fimbriae [154].
PROSPECTS
We have used two different secretion systems, the CU pilus
system and the T4SS, to contrast two classes of secretion systems,
those using two distinct machineries to release substrates first
through the inner membrane and next through the outer membrane
(CU pili), and those that elaborate a complex double membranespanning machinery to cross both membranes (T4SSs) in a single
step. In the two-step systems, the Sec translocon is used to
cross the inner membrane. No details on the Sec machinery
is provided in the present review as it has been excellently
documented elsewhere [28,29]. Subsequent transport through the
outer membrane involves two specialized proteins, a chaperone
and an usher. Both proteins are used to drive a process of
assembly and secretion, which is made energetically favourable
by the exquisite modulation of protein folding by the chaperone.
By maintaining the subunits in a semi-unfolded conformation,
the chaperone stores up energy which, when released upon DSE,
drives the pilus biogenesis process forward. Processes involving
folding energy to drive secretion are also at play in Type V
secretion, a secretion process through the outer membrane, which
requires a single transporter polypeptide forming a β-barrel in
the outer membrane. Type V secretion substrates, often (but not
always) are part of the same polypeptide that forms the β-barrel,
are driven through the barrel in an unfolded state, and folding
Two-step versus one-step secretion systems
post-transport is thought to drive the extrusion of the unfolded
peptide.
Because T4SSs are double membrane spanning, they have
access to the store of ATP in the cytoplasm and thus the
T4SS ATPases power secretion probably through complex,
undiscovered macromolecular relays regulating pore opening and
closure at both the inner and outer membranes. It will be one of
the challenges in the next few years to elucidate the molecular
basis of such relays. In this respect, the challenge extends to all
secretion systems, whether in bacteria or eukaryotes. It is likely
that within one class of secretion systems, common features will
be discovered, as well as distinct ones, but expanding knowledge
in this field is bound to provide inspiration for designing more
efficacious secretion systems inhibitors, which, at least in the
case of bacterial pathogens, are urgently needed.
Note added in proof (received 3 December 2009)
The structure of the outer membrane complex of the T4SS
encoded by the pKM101 plasmid has just been published [156].
This complex is made of the assembly of 14 copies of each of the
TraN/VirB7 protein, the C-terminal domain of the TraO/VirB9
protein and the C-terminal domain of the TraF/VirB10 protein.
This structure demonstrates unambiguously that (i) TraO/VirB9
does not form the outer membrane channel – instead, together
with TraN/VirB7, it forms a ring surrounding the TraF/VirB10
channel and buttressing the entire system against the inner leaflet
of the outer membrane, and (ii) the antennae of TraF/VirB10
forms the outer membrane channel and projects out across the
outer membrane.
FUNDING
Work in the authors’ laboratory was funded by the Wellcome Trust [grant number 082227
(to G.W.)]. A.T.R. was supported by the Fundação para a Ciência e Tecnologia [grant
number SFRH/BD/22254/2005].
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