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Biochimica et Biophysica Acta 1694 (2004) 219–234

Review

Type IV secretion: the Agrobacterium VirB/D4 and related

conjugation systems

Peter J. Christie*

Department of Microbiology and Molecular Genetics, UT-Houston Medical School, 6431 Fannin, JFB1.765, Houston TX 77030, USA

Received 2 December 2003; received in revised form 3 February 2004; accepted 3 February 2004

Available online 17 May 2004

Abstract

The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion

systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines

assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation

machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct

formation of stable contacts—the mating junction—between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a

nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating

junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA

trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Conjugation; Type IV secretion; DNA transfer; Pathogenesis; Protein translocation; Pilus; Coupling protein

1. Introduction VirB/D4 T4SS and related conjugation machines. In sum-

The type IV secretion systems (T4SS) translocate DNA

and protein substrates across the bacterial cell envelope.

These systems are classified on the basis of an ancestral

relatedness to bacterial conjugation machines [1]. Conse-

quently, members of this family include all conjugation

systems of Gram-negative and -positive bacteria and related

systems mediating DNA or protein translocation [2,3]. In

general, T4SS deliver their substrates to recipient cells via

direct cell-to-cell contact, although there are examples of

contact-independent protein export and DNA release to and

uptake from the extracellular milieu [1,4,5]. The nature of

type IV secretion substrates [6,7], the general architectural

features of various T4SS [2,3,8], and the cellular conse-

quences of macromolecular trafficking during pathogenesis

[1,7,9] have been the subjects of recent reviews.

Here, I will focus on the type IV machine itself, empha-

sizing the recent work on the Agrobacterium tumefaciens

0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbamcr.2004.02.013

* Tel: +1-713-500-5440; fax: +1-713-500-5499.

E-mail address: Peter.J.Christie@uth.tmc.edu (P.J. Christie).

marizing the recent progress, I hope it is evident that

detailed structure–function studies are advancing our state

of knowledge of conjugation and related systems extremely

rapidly. Within the next few years, a mechanistic under-

standing of these systems can be expected to approach that

of the general secretory pathway (GSP) and other systems

reviewed in this compendium.

2. The VirB/D4 T4SS operon structures and regulatory

controls

The A. tumefaciens VirB/D4 T4SS is encoded by the virB

and virD operons [10]. The virB operon encodes 11 genes,

virB1 through virB11. The virB gene products, termed the

mating pair formation (Mpf) proteins, elaborate a cell

envelope-spanning structure required for substrate transfer,

as well as an extracellular filament termed the T pilus that

mediates attachment to recipient cells [11]. The virD operon

encodes five genes, virD1 through virD5. virD1 and virD2

code for proteins that process the DNA substrate (T-DNA)

for transfer; these are termed the DNA transfer and replica-

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234220

tion (Dtr) proteins [12]. virD3 and virD5 code for proteins

that are nonessential for processing or transfer, and virD4

codes for the coupling protein (CP) [13,14]. The VirD4 CP

is not involved in T-DNA processing or biogenesis of the T

pilus but instead acts together with the Mpf structure to

deliver substrates across the cell envelope [10,11].

Type IV translocation systems whose subunits are related

to the virB and virD4 gene products are classified as the type

IVA subfamily [15]. The virB genes of the known T4SS

often comprise a single operon, whereas virD4 is usually

genetically linked to the genes involved in T-DNA process-

ing. In the systems studied to date, the T4SS genes are

activated in response to perception of environmental signals.

For example, phenolic and monosaccharide sugar molecules

released from wounded plant cells activate a VirA/VirG

two-component regulatory system which in turn induces

transcription of the A. tumefaciens virulence (vir) regulon

[13]. Additionally, low (1–5 mM) phosphate and an acidic

pH in the range of 5.0–5.5 are required for induction. A

PhoB-dependent regulatory system is thought to mediate the

response to phosphate starvation, whereas acid pH activates

transcription from an acid-inducible promoter upstream of

the virG response regulator gene and might also protonate

phenolic compounds leading to increased membrane per-

meability and VirA sensory perception [13,16]. Several

intracellular pathogens of mammals such as Brucella spp.

and Legionella pneumophila also induce expression of their

T4SS gene clusters in response to stress, e.g., to starvation

and acidic pH [17,18]. It is thus reasonable to predict that

plant and mammalian pathogens express T4SS genes and

elaborate these translocation systems in response to a

combination of specific inducing molecules, e.g., plant

phenolics, and more general environmental signals, e.g.,

acidic pH and low available nutrients, typically present at

sites of infection.

Focusing on the virB operon of A. tumefaciens, the

signals described above control transcription from the virB

promoter. There is also increasing evidence for the existence

of multiple transcriptional and posttranscriptional regulatory

controls that govern assembly of the VirB/D4 T4SS. At least

one internal promoter is thought to transcribe a subset of

virB genes in the absence of plant phenolic signals (L.

Banta, personal communication). Moreover, it was recently

shown that virB6 translation, but not production of a

functional VirB6 protein per se, is essential for efficient

synthesis of downstream virB gene products. The data

suggest virB6 translation might contribute to stabilization

of the distal portion of this polycistronic message [19].

Additionally, several closely juxtaposed or overlapping virB

genes, e.g., virB1/virB2/virB3/virB4 and virB7/virB8/virB9/

virB10, are probably translationally coupled, as supported

by results of genetic complementation studies [20]. Several

of these virB gene products mutually stabilize each other,

suggesting that co-translation serves to coordinate the tim-

ing and probably also the stoichiometric synthesis of inter-

acting subunits during machine biogenesis [19–23]. These

and other regulatory controls to be discovered probably

have evolved to maximize the chance and frequency of gene

transfer at an overall minimal energetic cost to the cell.

3. Type IV secretion substrates: processing and

recognition

3.1. Formation of the T-strand-relaxase intermediate

To understand the mechanistic details of type IV secre-

tion, it is first necessary to describe the secretion substrates

and associated processing reactions (Fig. 1). Early studies

showed that a complex of proteins assembled at the origin-

of-transfer (oriT ) sequence catalyzes a conversion of

supercoiled plasmid DNA to the open, relaxed form that

is detectable upon exposure to denaturing detergents [24].

This complex of processing proteins at oriT is termed the

relaxosome, and the subunit responsible for nicking at oriT

the relaxase. The relaxase is a transesterase that preserves

energy from cleavage of the phosphodiester backbone of

the strand destined for transfer (T-strand) as a stable

phosphotyrosyl intermediate [25]. Additionally, conjugal

DNA transfer proceeds in a 5V–3V direction [12], prompt-

ing a proposal that the relaxase covalently bound to the 5Vend of the T-strand supplies substrate recognition signals

and possibly also a piloting function to direct DNA

passage through the secretion channel [26,27]. Such a

model has now received very strong experimental support

(see below).

3.2. Protein substrates

Conjugation systems also export proteins independently

of DNA [28–34]. As shown in Fig. 1, these systems

translocate protein substrates, most probably in an unfolded

conformation, by chaperone-dependent or -independent

mechanisms. In this capacity, conjugation systems function-

ally resemble members of a second T4SS subfamily, the

‘effector translocators’. These systems are used by many

pathogens to deliver protein effectors to eukaryotic cells

during the course of infection [1]. Several assays have been

developed for identifying type IV protein substrates [6],

perhaps the most informative being the Cre/loxP recombi-

nation system [29,31,32]. As first demonstrated with the A.

tumefaciens VirB/D4 T4SS, protein substrates fused to the

Cre recombinase mediate transfer of Cre to target cells, as

shown by recombination at target loxP sites engineered into

target cell genomes [29,31,32]. In each case, transfer

requires the VirB/D4 T4SS as well as fusion of the recom-

binase to the N terminus of the protein substrate [29,31,32].

The question of whether relaxases are bona fide type IV

secretion substrates has been a subject of debate. Until

very recently, there was fairly convincing—albeit indi-

rect—evidence that the A. tumefaciens VirB/D4 T4SS

translocates the VirD2-T-strand and the plasmid RSF1010

Fig. 1. T4SS conjugally transfer DNA and protein substrates to target cells, as illustrated for the A. tumefaciens VirB/D4 T4SS. The transfer DNA (T-DNA) is

processed by Dtr proteins (VirD1, VirD2, and VirC1) bound at oriT-like border repeat sequences to form the VirD2-T-strand transfer intermediate. The VirB/D4

T4SS recruits the DNA transfer intermediate through binding of the VirD4 coupling protein (CP) to the positively charged C terminus of VirD2, which is

unfolded prior to export. The transfer intermediate is translocated across the cell envelope through a secretion channel composed of the VirD4 CP and the VirB

mating pair formation (Mpf) proteins; the precise route of translocation is undefined. Translocation of protein substrates similarly proceeds in three steps: (i)

processing, which is independent or dependent on chaperone binding to maintain substrate in an unfolded, nonaggregated conformation; (ii) recruitment,

mediated by CP binding to positively charged C-termini; and (iii) translocation, via the CP/Mpf secretion channel. IM, inner membrane; P, periplasm; OM,

outer membrane.

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234 221

MobA-T-strand intermediate to plant cells [26,27]. Now,

experiments using the Cre/loxP system have shown un-

equivocally that both the plasmid RP4 and L. pneumophila

Dot/Icm T4SS transfer a CreDMobARSF1010 fusion protein

to recipient E. coli and L. pneumophila cells, respectively

[34]. Of further interest, these T4SS were even capable of

translocating CreDrelaxase independently of a covalent

association with the T-strand transfer intermediate. With

the Cre/loxP recombination system, the investigators fur-

ther showed that the L. pneumophila Dot/Icm T4SS

delivers Cre fused to two other protein substrates, RalF

and LidA, to bacterial recipients, and they also identified a

large number of potential Dot/Icm substrates, designated

the Sid (Substrates of icm/dot) proteins. The Dot/Icm T4SS

was also shown to translocate one of these novel sub-

strates, SidC, to mammalian target cells [34].

In comparing the known protein substrates of conjuga-

tion systems and other T4SS, there are no obvious common

sequence motifs suggestive of a consensus secretion signal.

However, several findings now strongly indicate that such

signals reside at the C termini of type IV protein substrates

(Fig. 2). Again, the most compelling evidence derives from

use of the Cre/loxP system, whereby C-terminal fragments

of secretion substrates fused to Cre mediate T4SS-depen-

dent intercellular transfer of the recombinase. For example,

the A. tumefaciens VirB/D4 T4SS translocates Cre when

fused to C-terminal fragments of the VirE2, VirE3, and VirF

secretion substrates (Fig. 1) to plant and yeast cells

[29,31,32]. Similarly, the Dot/Icm T4SS translocates Cre

when fused to C-terminal fragments of the RalF and LidA

substrates to L. pneumophila recipients [34]. Upon close

examination, it is noteworthy that the C termini of A.

tumefaciens VirB/D4 T4SS substrates carry a conserved

Arg-X-Arg motif(s), whereas many T4SS substrates—in-

cluding relaxases from various conjugation systems—carry

an abundance of positively charged residues, mostly Arg,

within the last 30–50 residues. Interestingly, VirD3 also

possesses an abundance of basic residues at its C terminus

and thus might also be exported to plant cells during

infection, although this remains to be experimentally tested.

By contrast, the VirE1 secretion chaperone—which is not

recruited to the VirB/D4 T4SS [32,35]—does not carry such

a charge bias at its C terminus (Fig. 2).

How positive charge clusters contribute to type IV

substrate recognition is not known, but recently it was

shown that the C terminus mediates an interaction between

Fig. 2. Type IV secretion substrates contain clusters of positively charged residues at their C termini. This charge bias is a postulated feature of the type IV

secretion signal recognized by the coupling protein component of the T4SS [31,33,35]. Shown are the C-terminal residues, with Arg (R), Lys (K), and His (H)

residues highlighted with box shading and the Arg-X-Arg motifs of VirB/D4 T4SS substrates with wavy underlines. Known or potential substrates include

VirB/D4 T4SS substrates encoded by the pTiC58 (C58) and pTiA6NC (A6) plasmids of A. tumefaciens, relaxases encoded by the plasmids listed, CagA

exported by the H. pylori Cag T4SS, and RalF exported by the L. pneumophila Dot/Icm T4SS. VirD3 proteins from the C58 and A6 plasmids are candidate

VirB/D4 secretion substrates on the basis of a C-terminal charge bias. VirE1 is not predicted to be a substrate on the basis of charge distribution, in agreement

with findings that this secretion chaperone is not recruited by the VirD4 CP to the VirB/D4 T4SS [32,35].

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234222

the VirE2 secretion substrate and the VirD4 CP of the VirB/

D4 T4SS. Following up a report that VirD4 localizes at the

cell poles of A. tumefaciens [36], cells were engineered to

produce a GFP-VirE2 fusion protein and assayed for VirD4-

dependent recruitment of the fusion protein to the poles.

Indeed, cells displayed polar fluorescence when producing

GFP fused to the N terminus, but not to the C terminus, of

the substrate (Fig. 3) [35]. Two additional cytological

assays, termed cytology two-hybrid (C2H) and bimolecular

fluorescence complementation (BiFC), as well as a coupled

chemical-cross-linking and immunoprecipitation assay sup-

plied further evidence for the VirD4–VirE2 interaction. By

use of BiFC, it was further shown that the C-terminal 100

residues of VirE2 are necessary and sufficient for recruit-

Fig. 3. The A. tumefaciens T4SS and a secretion substrate localize at the cell

poles. The T pili localize at cell poles as detected by electron microscopy

(kindly provided by Alain Bernadac), the VirD4 CP localizes at the cell

poles as shown by polar fluorescence of a VirD4-GFP fusion protein, and

the VirD4 CP recruits VirE2 to the cell poles as shown by VirD4-dependent

polar fluorescence of GFP-VirE2. GFP itself distributes throughout the

cytoplasm of A. tumefaciens cells (middle panel) (see Ref. [35]).

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234 223

ment of GFP-VirE2 derivatives to VirD4. Although attempts

to further delineate the N-terminal boundary of the putative

secretion signal were unsuccessful due to problems of

protein instability, a small deletion of the extreme C

terminus as well as a peptide insertion near the Arg cluster

impeded complex formation [35]. These findings, together

with the demonstration that a VirE2 C-terminal fragment

directs Cre translocation, suggest that the charged C termini

of type IV secretion substrates probably contribute to

substrate recognition by mediating productive contacts with

the CP component of the T4SS (Fig. 1).

All conjugation systems—at least of Gram-negative

bacteria—possess a CP and therefore can be predicted to

export substrates via CP-dependent recognition of C-termi-

nal signals. A few members of the effector translocator

subfamily do not possess a recognizable CP subunit [1]. Of

these, the Bordetella pertussis Ptl T4SS is the only system

for which a substrate, pertussis toxin (PT), is known. For

secretion of PT subunits across the IM, B. pertussis employs

the GSP, and consequently these subunits carry characteris-

tic GSP secretion signals at their N termini. Upon delivery

across the IM, the subunits assemble as the PT holotoxin for

delivery by the Ptl T4SS across the OM. How PT holotoxin

is recognized in the periplasm by the Ptl system is not

known [37].

4. Initiation of T-strand transfer: substrate recruitment

by the coupling protein

By the mid-1990s it was well-established that a given

T4SS, e.g., encoded by plasmids R388 (IncW) or RP4

(IncPa) or the A. tumefaciens VirB/D4 system, translocates

a restricted set of substrates that includes the cognate plasmid

or oncogenic T-DNA, one or a few mobilizable plasmids

such as ColE1 or RSF1010, and one or a few proteins [12].

The mobilizable plasmids carry oriT sequences and the Mob

processing proteins acting at oriT, but lack the genes for their

own T4SS. To identify machine components involved in

substrate recognition, several investigators constructed chi-

meric systems composed of a CP from one T4SS and an Mpf

structure from a second T4SS. Such chimeric T4SS were

shown to be functional, and, furthermore, these systems

exported substrates characteristically translocated by the

T4SS from which the CP was derived. For example, TraG

from plasmid RP4 substituted for TrwB of plasmid R388—

and vice versa—with respect to RSF1010 mobilization. Yet,

the chimeric TraGRP4/MpfR388 system failed to transfer

R388, and the TrwBR388/MpfRP4 system failed to transfer

RP4. Similarly, both VirD4 and the Ti plasmid TraG CPs of

A. tumefaciens substituted for TraGRP4 with respect to

RSF1010 mobilization, but not transfer of RP4 [14,38–

40]. These findings strongly suggested that the CP links

the Dtr processing proteins bound at oriT—the relaxo-

some—to the T4SS, hence the origin of the term ‘coupling

protein’.

A couple of CPs were then purified and shown to bind

DNA in vitro [41–43]. These DNA-binding studies dem-

onstrated that single-stranded (ss) DNA is preferred over

double-stranded (ds) DNA as expected, yet binding was also

sequence nonspecific. Thus, it was proposed that the CP

recognizes DNA substrates by virtue of interactions with the

relaxase and other relaxosomal subunits bound at oriT—not

the ssDNA intermediate per se. Indeed, in vitro studies have

now demonstrated several CP–relaxase interactions. The

TraGRP4 CP interacts with TraIRP4 relaxase/helicase [43]

and with Mob relaxase of the mobilizable plasmid pBHR1

[44]. The TrwBR388 CP interacts with TrwCR388 relaxase/

helicase, and the TraDF CP interacts with TraMF, a Dtr

protein that participates in processing by binding at sites

near the F plasmid oriT sequence [45]. As noted above,

several relaxases possess an abundance of positively

charged residues at their C termini (Fig. 2); these residues

likely mediate productive contacts with the CP. However, in

view of the fact that a given T4SS translocates a restricted

set of substrates, it is also likely that other C-terminal

signals, e.g., a structural motif presented by folding of the

C-terminal domain, probably also contributes to substrate

selection.

Upon recruitment, how does the CP mediate the next

step(s) of transfer? Recent structural studies have begun to

shed light on the answer to this question. Topology studies

have shown that CPs are composed of an N-proximal region

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234224

that includes two transmembrane helices and a small peri-

plasmic domain, and a large C-terminal region that resides in

the cytoplasm (Fig. 4) [46,47]. Very interestingly, a crystal

structure solved for the soluble domain of TrwBR388

(TrwBDN70) showed that six equivalent protomers form a

spherical particle of overall dimensions 110 A in diameter

and 90 A in height. This ring-like structure possesses a

central channel of 20 A in diameter, constricted to 8 A at the

entrance facing the cytoplasm. This channel traverses the

structure, possibly connecting cytoplasm with periplasm

[48,49]. An appendix corresponding to the N-terminal TM

domain was discernible by image averaging of electron

micrographs [42]. This overall structure bears a striking

resemblance to the F1-ATPase a3h3 heterohexamer, whereas

the structure of the soluble domain closely resembles DNA

ring helicases and other proteins that translocate along ss- or

ds-DNA (Fig. 4) [50].

The oligomeric state of CPs in solution was also char-

acterized and results suggest the situation in vivo is prob-

ably more complex than was revealed by the X-ray crystal

Fig. 4. Topologies and oligomeric structures of VirB/D4 T4SS components. The

ATP binding; the VirB11 ATPase undergoes dramatic conformational changes in its

also postulated to bind and hydrolyze ATP. These components plus the channel sub

A disulfide-linked dimer of VirB7 lipoprotein and secretin-like VirB9 are postula

Independently of the VirD4 CP, the VirB proteins direct polymerization of the T pi

inner membrane; P, periplasm; OM, outer membrane.

structure. For example, the TrwBDN70 purified as a mono-

mer, raising the possibility that the hexamer identified by

crystallography derived from the precipitation conditions

employed during crystallization, e.g., high protein concen-

tration and dehydration. Moreover, the full-length protein

containing the N-terminal transmembrane stem purified

both as a monomer and as a hexamer [42]. This finding

led to a proposal that this CP undergoes monomer–hexamer

transitions in vivo, possibly mediated by nucleotide and/or

substrate binding (Fig. 4). Such dynamic structural changes

might play an important role in substrate translocation. In

other studies, purified full-length TraGRP4 and TraDF

formed large multimers of >15 subunits; these were prob-

ably aggregates as indicated by their broad sedimentation

profiles in glycerol gradients [43]. By contrast, HP0524Cagdeleted of its N-terminal membrane-spanning domain puri-

fied as a multimer of ~4–5 subunits, whereas corresponding

TraGRP4 and TrwBR388 truncation derivatives lacking the N-

terminal transmembrane domain purified exclusively as

monomers [43,51]. Together, these findings point to the

VirD4 CP undergoes monomer–hexamer transitions possibly mediated by

N-terminal domain as a function of ATP binding and ADP release; VirB4 is

units shown are postulated to form a membrane-spanning secretion channel.

ted to form a pore at the OM for pilus protusion and/or substrate transfer.

lus, which may or may not be physically joined to the secretion channel. IM,

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234 225

importance of the N-terminal transmembrane domain for CP

oligomerization in vivo, whereas its presence at least for

some CPs seems to induce aggregation in vitro.

In addition to a role in oligomerization, the N-terminal

region of A. tumefaciens VirD4 directs localization of the

CP predominantly to the cell poles [36]. A deletion muta-

genesis study established that residues in the periplasmic

loop contribute to polar positioning. This VirD4 localiza-

tion and an observation that the T pilus is polar-localized

(Fig. 3) [11] suggest that this T4SS localizes at the cell

poles. Interestingly, however, a few years ago it was

reported that some VirB proteins, notably VirB8, VirB9,

and VirB10, form foci around the cell periphery [52]. Since

then, similar types of foci with the TrhC (VirB4 homolog)

subunit of the plasmid R27 T4SS were observed [53]. Such

foci might correspond to artifacts from protein overproduc-

tion, but it is also possible that such complexes represent

physiologically relevant intermediate- or even terminal-

pathway structures.

Recently, the nature of CP interactions with Mpf subunits

has been explored. TraG of plasmid R27 from Salmonella

enterica was found to interact with the VirB10-like protein,

TrhBR27 [54]. As discussed below, the VirB10-like proteins

are probably structural scaffolds required for assembly of

the transenvelope T4SS. As expected from their membrane

topologies, the TraGR27 CP interacted with the N-terminal

region of TrhBR27 bearing a small cytoplasmic domain, a

TM, and a portion of the periplasmic region. A second study

further showed that the N-terminal domains of both the

TrwBR388 CP and the VirB10-like TrwER388 protein mediate

their interaction [55]. Consistent with the results obtained

with the chimeric T4SS described above, this study also

showed that CPs from three different T4SS interacted with

the VirB10-like proteins of the cognate system as well as

other systems. Additionally, TrwBR388 CP was shown to

interact with the TrwCR388 relaxase, establishing a direct

link between the relaxosome and the Mpf structure [55].

In summary, the CP recruits DNA substrates to the

secretion apparatus through contacts with the DNA-process-

ing proteins (Figs. 1 and 4). Then, through the VirB10 and

perhaps other contacts, the CP coordinates passage of the T-

strand through the Mpf channel. Precisely how this latter

reaction occurs is not known, but the CP structures suggest

one possibility—the CP acts as a translocase to deliver the

substrate across the inner membrane. In this context, it is

interesting to note that CPs share limited sequence similar-

ities with two DNA translocases, Bacillus subtilis SpoIIIE

and E. coli FtsK [56,57].

5. Evidence for a transenvelope secretion channel: the

Mpf structure

All of the VirB proteins except for VirB1 are required for

substrate export [20]. Surprisingly, however, high-resolution

electron microscopy studies of mating cells, e.g., RP4-

carrying donor and recipient mating pairs, thus far have

not identified a supramolecular organelle at cellular junc-

tions. Instead, an electron dense layer was seen between

tightly juxtaposed donor and recipient cell surfaces [58,59].

Junctions at the poles were estimated to be f 150–200 nm

in length, whereas those along the cell body extended up to

1500 nm in length. No T4SS organelles reminiscent of the

basal body of flagella or needle complexes of type III

secretion systems (T3SS) were evident. Corresponding

fractionation studies of several conjugation systems have

shown that most subunits co-partition with both membranes

in sucrose gradients and/or with a membrane fraction whose

density is intermediate between those of the IM and OM

[60–63]. The T4SS might thus assemble at zones of

membrane adhesion, which in turn could correspond to

the sites of electron density when mating pairs are examined

by electron microscopy [59].

Despite a lack of ultrastructural resolution, several find-

ings support the existence of an envelope-spanning secre-

tory apparatus. For example, the presumptive VirB/D4

mating channel or subcomplexes thereof can be isolated

by membrane solubilization with nonionic detergents, e.g.,

dodecyl maltoside, octylglucoside, or Triton X-100. In one

study, high-molecular-weight VirB-containing species were

detected by blue native gel electrophoresis and size exclu-

sion chromatography. At least two large complexes were

identified, one composed of the pilus-associated proteins

VirB2, VirB5, and VirB7, and the second composed of

several other VirB proteins and VirD4 [64]. Independent

studies have also reported the isolation of a VirB2/VirB5/

VirB7 complex and subcomplexes of other VirB proteins,

e.g., VirB7/VirB9, VirB6/VirB7/VirB9 and VirB7/VirB9/

VirB10 by detergent-solubilization and immunoprecipita-

tion or GST-pull-down assays [19,22,23]. Results from

two-hybrid screens complement these findings and further

suggest the possibility of other pairwise interactions in vivo

[52,65]. Similar studies of the F-like [2,63] and RP4 [62]

have identified presumptive complexes among putative

channel components.

Taken together with computer-based predictions and

topology studies of individual VirB subunits, a general

architecture for the VirB/D4 T4SS can be presented (Figs.

4 and 5). Accordingly, the VirD4 CP and the two Mpf

ATPases, VirB4 and VirB11 ATPase, are positioned pre-

dominantly or exclusively at the cytoplasmic face of the IM.

VirB6 is a highly hydrophobic protein predicted to span the

IM several times, whereas VirB8 and VirB10 are bitopic

proteins with short N-terminal cytoplasmic domains, a TM,

and large C-terminal periplasmic domains. VirB2, VirB3,

and VirB5 are located in the periplasm, and VirB2, VirB5,

and VirB7 also assemble as the extracellular T pilus. VirB7,

a small lipoprotein, and VirB9 form a covalently cross-

linked dimer and this dimer or more probably a higher-order

VirB7–VirB9 multimer assembles at the OM. Among the

VirB proteins, VirB9 is the best candidate for forming an

oligomeric secretion-like pore for mediating passage of

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234226

substrates and/or protrusion of the T pilus across the OM

(Fig. 4).

6. Energetic components—VirB4 and VirB11

In the next sections, information will be presented about

the putative VirB channel subunits and the genetic require-

ments for substrate passage, focusing first on the energetic

components.

VirB11: VirB11 is a member of a large family of ATPases

associated with systems dedicated to secretion of macro-

molecules [66]. Homologs are widely distributed among the

Gram-negative bacteria, and they also function in secretion

systems of Gram-positive bacteria as well as various species

of the Archaea.

VirB11 itself is extremely insoluble and has been difficult

to characterize in vitro, whereas soluble forms of VirB11

homologs have been characterized enzymatically and struc-

turally. Purified TrbBRP4, TrwCR388, and H. pylori

HP0525Cag were each shown to assemble as homohexa-

meric rings discernible by electron microscopy [67]. Like

the TrwBR388 CP, HP0525Cag also presented as a homohex-

amer by X-ray crystallography [68]. However, quite unlike

the CP, HP0525Cag is a double-stacked ring with a central

cavity of f50 A in diameter. One ring is formed by N-

terminal domain interactions, the second by C-terminal

domain interactions. The N-terminal domain is unique to

HP0525Cag, whereas the C-terminal domain adopts a RecA

fold. In the first crystal structure generated, a molecule of

ADP is bound at the interface between the two domains

[68].

The overall HP0525Cag structure appears to be highly

conserved, even among distantly related ATPases associated

with other transport or fimbrial biogenesis systems. For

example, L. pneumophila DotB of the Dot/Icm T4SS [69],

Aquifex aeolicus PilT associated with type IV pilus retrac-

tion (these pili are assembled via a type II secretion system

(T2SS) and are not related to type IV secretion) [70], and

Actinobacillus actinomycetemcomitans TadA associated

with Flp pilus biogenesis [71]; each purifies as ring-shaped

homohexamers with dimensions similar to those observed

for HP0525Cag. A crystal structure of Vibrio cholerae EpsE

associated with the Eps T2SS recently was reported [72],

and the closest structural homolog is HP0525Cag.

Both EpsEVch and HP0525Cag also are structurally similar

to members of the AAA ATPase superfamily [72,73]. These

structural similarities are intriguing because AAA ATPases

display functions compatible with the possible activities of

the EpsEVch and VirB11-like proteins. Many AAA ATPases

are energy-dependent unfoldases whose activities are asso-

ciated with substrate remodeling. In view of the fact that the

T4SS are protein exporters, the VirB11-like ATPases might

act to unfold these substrates at the channel entrance.

Alternatively or in addition, a VirB11 chaperone activity

might facilitate assembly of the secretory apparatus and/or

the T pilus. Another interesting structural relationship was

noted with the p97 AAA ATPase, which is involved in

membrane fusion and organelle biogenesis [74,75]. In this

context, it is interesting that the VirB11 homolog, TrwDR388,

was shown to induce dose-dependent vesicle aggregation

and intervesicular mixing of outer monolayer lipids [76,77].

While the investigators suggest this activity might be related

to early stages of transporter assembly, it also seems possible

that VirB11-like proteins are involved in membrane remod-

eling during formation of the mating pair junction.

As with TrwBR388 CP, the X-ray structure of the ADP-

bound form of HP0525Cag offers only a snapshot view of

the VirB11 ATPase structure. More recent comparative

studies of the apo-, non-hydrolyzable ATPgS-, and ADP-

bound structures, together with analytical ultracentrifugation

experiments, showed that one consequence of ATP-binding

is a swiveling of the N-terminal domains [75]. This confor-

mational switch converts the open and asymmetric hexamer

to a more compact, symmetrical hexamer (Fig. 4). More-

over, a molecule of polyethylene glycol was found bound to

the N-terminal domain in the HP0525Cag crystal structure

[68]. In vivo, this is probably a molecule of phospholipid,

consistent with results of fractionation studies and other

experimental findings showing that the VirB11-like

ATPases associate tightly but peripherally at the inner face

of the IM [78,79]. Interestingly, VirB11 as well as DotBLpn

mutants with Walker A nucleotide-binding motif defects

bind the IM more tightly than the wild-type protein, sug-

gestive of an ATP-regulated membrane interaction [69,79].

Finally, genetic studies supplied evidence for the coor-

dinated actions of VirB11 and the VirD4 CP for substrate

transfer. A genetic screen resulted in isolation of virB11

alleles displaying dominance in virB11/virB11* merodiploid

strains [80]. These alleles were then subclassified as func-

tional or nonfunctional on the basis of whether the mutant

protein supports substrate transfer in the absence of native

VirB11. Studies of the functional subclass identified one

mutant protein that supported substrate transfer (Tra+) but

completely blocked biogenesis of the T pilus (Pil�). Con-

versely, some mutant proteins of the nonfunctional class

supported T pilus biogenesis but blocked substrate transfer

(Tra�, Pil+). Hence, these were termed ‘‘uncoupling’’ muta-

tions, and their isolation indicates that VirB11 contributes

both to pilus biogenesis and to assembly or function of the

secretion machine by mechanisms distinguishable by muta-

tion [81]. Such mutations might selectively disrupt VirB11

contacts with VirD4 to block substrate transfer or, alterna-

tively, contacts with other Mpf proteins to disrupt either

transfer or pilus biogenesis.

In considering the nature of the structural interaction

between the CP and VirB11-like protein hexamers, in

principle, the two subunits could form a composite dou-

ble-stacked ring at the IM. Yet, such a structure is difficult

to envision in view of the dimensions of the hexameric

structures of the respective homologs and the subcellular

dispositions of both subunits, e.g., both homohexamers

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234 227

associate with the IM, one integrally and one peripherally

(Fig. 4). A more likely model is that the VirD4 and

VirB11 hexamers form independently but localize next to

each other at the IM (Fig. 5). These structures would

undergo coordinated ATP-dependent conformational

switches to mediate substrate recruitment to the CP and

delivery to VirB11 for subsequent transfer through the

Mpf structure. Consistent with this structural arrangement,

Llosa et al. [57] developed a model based on evolutionary

considerations that depicts conjugation systems as two

separately acting IM translocases: the CP evolved from

ring helicases exclusively for T-strand export and the Mpf

proteins correspond to an ancestral protein export system.

According to this model, the CP still functions as a

general recruitment factor for all T4SS substrates. Instead

of delivering the entire substrate to the Mpf proteins,

however, the CP would translocate the T-strand across

the IM while simultaneously transferring the relaxase to

VirB11 and the other Mpf proteins for secretion. This

interesting model satisfactorily explains many experimen-

tal findings. But without invoking significant, dynamic

Fig. 5. A model depicting the A. tumefaciens VirB/D4 T4SS as a single,

supramolecular organelle. The VirD4 CP is a homomultimeric integral

membrane complex required for substrate transfer. The VirB proteins

assemble as a secretion channel and an extracellular T pilus. VirD4 and the

VirB proteins function together to mediate substrate transfer, and the VirB

proteins direct pilus assembly. Cells are postulated to shed the adhesive T

pilus as a means of promoting aggregation of donor and recipients on solid

surfaces. IM, inner membrane; P, periplasm; OM, outer membrane.

and tightly coordinated changes in CP/VirB11 conforma-

tions and oligomeric structures, one still has difficulty

envisioning how two translocases sitting next to one

another at the membrane coordinate their activities to

drive secretion of one substrate—the covalently bound

nucleoprotein particle—across the IM.

VirB4: VirB4 possesses two putative membrane-spanning

domains, one near the N terminus and a second more

centrally located and near the Walker A nucleoside triphos-

phate binding motif (Fig. 4) [78]. A transmembrane topol-

ogy is also predicted for VirB4 homologs, e.g., TrbCRP4

[82]. With respect to subunit–subunit contacts, to date, there

is biochemical evidence only for a VirB4 self-interaction

[83]. A VirB4–VirB11 interaction is suggested by a yeast

two-hybrid screen, but not yet confirmed with complemen-

tary biochemical or genetic approaches [65].

ATP hydrolysis has not been convincingly shown for

purified VirB4-type proteins, but these proteins require

intact Walker A motifs to mediate substrate export

[82,84]. Intriguingly, several years ago it was reported that

the presence of a subset of VirB proteins in agrobacterial

recipient cells leads to a significant increase in the efficiency

of plasmid acquisition in matings with agrobacterial donors

[85]. These findings led to a proposal that these VirB

proteins assemble as a complex that stabilizes mating

junctions or perhaps directly facilitate DNA uptake across

the recipient cell envelope. VirB4 is one of the VirB proteins

that contribute to this stimulatory effect [19]. However, it

was also shown that a Walker A mutation does not alter the

capacity of VirB4 to stimulate plasmid acquisition by

recipients. Therefore, VirB4 probably contributes structural

information necessary for substrate transfer—in either di-

rection—across the cell envelope, but an intact ATP-binding

domain is necessary for configuring this T4SS specifically

for substrate export [83]. Clearly, the VirD4, VirB11, and

VirB4 proteins must interact in complex and dynamic

ways—most probably via ATP-driven conformational

changes—to energize substrate transfer to and across the

IM (Figs. 4 and 5).

7. IM proteins—VirB6, VirB8, and VirB10

In contrast to the phylogenetically wide distribution of

the VirB11-like ATPases, the VirB6, VirB8, and VirB10 IM

proteins or functional orthologs are found mainly as com-

ponents of T4SS elaborated by Gram-negative bacteria

[1,2,66].

VirB6: VirB6 is a highly hydrophobic protein with five,

six or nine possible TMs predicted by the TmPred,

TMHMM, or TopPred2 algorithm, respectively (Fig. 4).

Polytopic proteins are common components of secretion

or transport systems, e.g., SecY, SecE, and SecG of the GSP

[86] and TatC of the twin-arginine translocation (TAT)

system [87]. Thus, early on it was postulated that VirB6 is

a channel constituent. A virB6 deletion mutation results in

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234228

diminished levels of several VirB proteins, consistent with a

role in maintaining the structural integrity of this T4SS

[20,22,88]. Additionally, Krall et al. [64] showed that VirB6

co-fractionates with other VirB proteins as a high-molecu-

lar-weight complex(es) in detergent-solubilized cell extracts,

further suggestive of interactions with other VirB proteins as

a stable supramolecular structure. Protein–protein interac-

tion studies also have supplied evidence for VirB6 contacts

with the VirB7 and VirB9 OM proteins. These interactions

influence the formation of a disulfide cross-linked VirB7

homodimer as well as higher-order VirB9 multimers detect-

able by electrophoresis through protein gels under nonre-

ducing conditions. Moreover, several four-residue insertion

mutations affecting VirB9 multimerization mapped to a

central, periplasmic loop of VirB6. The VirB6 interaction

with the VirB7 and VirB9 proteins might constitute a

contact between IM and OM channel subunits of this

T4SS (see below) [22].

VirB8 and VirB10: VirB8 and VirB10 are both bitopic

IM proteins with N-terminal proximal TMs and large C-

terminal periplasmic domains (Fig. 4) [46]. VirB8 and

VirB10 each were shown to self-associate and also to

interact with each other and with VirB9 [23,52,89]. Muta-

tions affecting these interactions abolished virulence, con-

firming that VirB8, VirB9, VirB10 complex formation is

essential for machine function [52,89]. As noted above,

foci composed of VirB8, VirB9, and VirB10 were identi-

fied around the cell periphery by immunofluorescence and

electron microscopy [89]. On examination of various A.

tumefaciens mutants, it was further shown that VirB8

production is necessary for focal formation of VirB9 and

VirB10. The investigators proposed that VirB8 nucleates

assembly of the VirB8, VirB9, VirB10 complex [89].

Consistent with the notion that these proteins form a stable

complex, these three proteins co-fractionate in blue native

gels and during gel filtration [64]. These findings plus the

demonstrations of interactions between homologs of

VirB10 and VirD4 [54,55] suggest that the VirB10 proteins

function as structural scaffolds, linking CPs to their cog-

nate T4SS and also IM subcomplexes to OM subcom-

plexes of the T4SS. Consistent with such a transmembrane

‘bridging’ function, VirB10 proteins possess Pro- or Gly-

rich central domains suggestive of a rigid, extended struc-

ture in the periplasm.

8. Periplasmic/OM proteins—VirB2, VirB7 and VirB9,

VirB3 and VirB5, VirB1 and AcvB/VirJ

Six VirB proteins—VirB1, VirB2, VirB3, VirB5, VirB7,

and VirB9—carry characteristic GSP signal sequences at

their N termini. All of these proteins localize predominantly

in the periplasm or OM, whereas VirB2, VirB5, and VirB7

also polymerize to form the extracellular T pilus (Fig. 4).

The contributions of the cell-associated forms of these

proteins to substrate transfer are discussed below.

VirB2: VirB2 is the pilin subunit of the T pilus (Fig. 4)

[11]. The overall pilus biogenesis pathway is poorly under-

stood (see below), but several of the processing reactions

leading to mature pilin are well characterized for this and

other conjugation systems. The propilin is delivered into or

across the IM by the GSP. In the periplasm, cleavage of the

signal sequence, most probably by leader peptidase LepB, is

followed by one or more modification reactions. For exam-

ple, the F TraA-like pilins are N-terminally acetylated, RP4

TrbCRP4 is proteolytically cleaved near its C terminus, and

both TrbCRP4 and A. tumefaciens VirB2 undergo a novel

head-to-tail cyclization reaction that covalently links N- and

C-terminal residues making a closed circular polypeptide

[2,90–93]. In all systems, mature pilin is integrated into the

inner membrane; for F TraA, integration requires TraQ [2].

The integrated pilin is thought to comprise a pool of pilin

monomers accessible for polymerization as the T pilus upon

receipt of an exogenous signal from recipient cells.

The cell-associated VirB2 might be used exclusively for

pilus biogenesis on demand, but one line of study suggests

at least a portion of the pilin alternatively assembles as a

structural subunit of the secretion channel in the periplasm

(Fig. 4). We asked whether the Tra+, Pil� mutant strains

described above are still capable of translocating substrate

in the absence of the VirB2 pilin protein. We deleted the

virB2 gene from strains synthesizing the virB9 or virB11

‘‘uncoupling’’ alleles, and then we tested whether such Pil�

cells retained the capacity to translocate substrates. The

virB2 gene deletion abolished substrate transfer, establish-

ing that VirB2 pilin is essential for substrate transfer (S.J.

Jakubowski, unpublished data). Thus, the VirB/D4 T4SS

can transfer DNA intercellularly in the absence of detectable

T pilus, but not in the absence of pilin protein. The simplest

explanation for these findings is that the pilin protein is a

structural subunit of the secretion channel in the periplasm

or at the OM.

VirB3 and VirB5: VirB3 and VirB5 are required for

substrate transfer. Both proteins are exported across the

IM where they sort to the periplasm and the OM (Fig. 4).

Both proteins also are thought to play a role in biogenesis of

the T pilus and, indeed, VirB5 has been shown to interact

with the cell-associated VirB2 as well as with the extracel-

lular T pilus [64,93,94]. With respect to VirB3, presently,

there are no reports of interactions with other VirB proteins,

but a finding that production of VirB6 enhances steady-state

levels of VirB5 and VirB3 suggests the possibility of

stabilizing interactions among these VirB proteins [88].

Both proteins might thus contribute to assembly or function

of the secretion channel in the periplasm.

Very recently, a crystal structure of a VirB5 homolog,

TraC of pKM101, was presented [95]. This is a single

domain protein with a mostly a-helical, elongated structure.

Analyses of TraCKM101 mutants identified regions likely to

be involved in protein–protein interactions. A couple of

mutant proteins conferred altered conjugation frequencies as

well as defects in infection by bacteriophages PRD1 and

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234 229

Ike. The investigators have proposed that the VirB5-like

proteins bind along the pilus, including the pilus tip (the site

of Ike attachment) and base (the site of PRD1 attachment);

these sites of contact are thought to promote pilus biogen-

esis as well as adherence to recipient cells (Figs. 4 and 5)

[95]. At this point, however, the TraCKM101 structure does

not provide much insight into how this family of pilus-

associated proteins contributes to channel formation or

activity. One possibility is that VirB5 interacts with cell-

associated VirB2 pilin in the periplasm and modulates

VirB2 interactions with the VirB9 OM protein or the VirB6,

VirB8, and VirB10 proteins in the periplasm or at the IM.

VirB7 and VirB9: VirB9 has nine possible h-sheet OM-

spanning segments according to the Schirmer-Cowan algo-

rithm [96] (S.J. Jakubowski, unpublished data). VirB9

assembles as a disulfide cross-linked heterodimer with the

VirB7 lipoprotein (Fig. 4) [97–99]. Consistent with an OM

localization of this dimer, the VirB7 sequence at the N-

terminal lipid-modification site (CQTN) is devoid of the

Asp2 + IM-retention signal [100]. Mutations of the reactive

Cys residues involved in disulfide bridge formation desta-

bilize VirB7 and VirB9 and several other VirB proteins, and

confer avirulence unless VirB9 is significantly overpro-

duced [96–99,101]. Thus, the heterodimer or, more proba-

bly, a higher-order multimer of dimers represents the

physiologically relevant form of these subunits.

VirB9 also has sequence similarities with pore-forming

domains of secretin proteins associated with type II (PulD)

[102] and III (YscC) [103] secretion, filamentous bacterio-

phage f1 (pIV) [104], and Pap pilus biogenesis (PapC) [105]

[2; S.J. Jakubowski, unpublished data]. Intriguing parallels

also exist with a subset of secretins, namely those that form

a stabilizing interaction with a cognate OM lipoprotein (e.g.,

PulS–PulD) [102]. These findings suggest the interesting

possibility that VirB9, with a structural contribution of

VirB7 lipoprotein, assembles as a secretin for substrate

passage and, possibly, protrusion of the T pilus across the

OM. At this time, however, a secretin-like function remains

an enticing, but experimentally unproven, model.

VirB7 also forms a disulfide cross-linked homodimer that

co-partitions with the IM and OM fractions and, curiously,

is released into the extracellular milieu at abundant levels

[61,90]. In pilus-producing cells, the VirB7 homodimer

associates with T pili that are released by sloughing or

shearing from the cell surface (Fig. 4) [90]. In pilus-minus

cells, the lipoprotein is also found in this extracellular

fraction. The physiological relevance of the cell-bound

and extracellular VirB7 homodimer is not known, but these

novel findings suggest this small lipoprotein contributes in

several ways to assembly or function of this T4SS.

VirB1 and AcvB/VirJ: Three periplasmic proteins were

postulated to contribute to T-strand transfer through effects

on T4SS biogenesis or function. VirB1 carries a trans-

glycosylase motif and is thought to ‘‘punch’’ holes in the

peptidoglycan to facilitate T4SS assembly [106]. VirB1-

related transglycosylases contribute to conjugal DNA trans-

fer as most extensively shown for the P19 transglycosylase

of plasmid R1 [107]. Additionally, transglycosylases are

important for assembly of other T4SS, PtlE for B. pertussis

Ptl [108], and AtlA for an F-like Tra system carried on the

gonococcal genetic island of N. gonorrhoeae that mediates

DNA release into the milieu [4]. VirB1 transglycosylase

activity is important for biogenesis of the A. tumefaciens T

pilus, but not for substrate transfer, as shown by the capacity

of the DvirB1 mutant to incite tumor formation on plants

[19].

The Ti plasmid-encoded VirJ protein and its chromo-

somal homolog, AcvB, were shown to localize in the

periplasm [109] and evidence was also presented for a VirJ

interaction with the VirB/D4 T4SS as well as the VirE2

effector and VirD2 relaxase [110]. A virJ/acvB mutant is

avirulent [109], leading to a proposal that these proteins

function as periplasmic chaperones to mediate substrate

transfer through the periplasm [110]. At this time, however,

it is premature to conclude that AcvB and VirJ contribute

directly to substrate transfer through the VirB/D4 T4SS.

9. T pilus and secretion channel: anatomical and spatial

relationships

The genetic requirements for assembly of the secretion

channel closely match those for T pilus biogenesis (Fig. 4).

All of the VirB proteins are needed for both processes, with

the exception of VirB1 transglycosylase, which as noted

above is required for pilus production but not for substrate

transfer. Thus, it is tempting to depict channel assembly and

pilus outgrowth as two interlocked reaction pathways that

culminate in assembly of a single, supramolecular organelle,

the secretion channel/T-pilus. Accordingly, such a structure

would consist of a set of IM channel proteins and a pilus

structure extending through the periplasm and a (VirB9?)

secretin-like pore at the OM (Fig. 5). Consistent with such a

model, in other conjugation systems it has been shown that

pilin subunits are recruited from the IM and added to the

base of the pilus [2].

A prediction of this model is that the lumen of the pilus

serves as a conduit for type IV secretion substrates. In fact,

however, there is no strong experimental support for sub-

strate transfer through an intact T pilus. To the contrary, at

least two lines of evidence argue strongly against the

conduit model, at least for the VirB/D4 system. First, a

nonpolar null mutation of virB1 fails to assemble the T

pilus, yet the DvirB1 mutant still translocates the oncogenic

T-DNA to plant cells. This shows that the secretory appa-

ratus is intact in Pil� cells [20]. Second, we have now

isolated ‘‘uncoupling’’ mutations in several VirB proteins—

VirB11 [10], VirB6 [22], and VirB9 (S.J. Jakubowski,

unpublished data). As with the DvirB1 derivative, strains

producing these mutant proteins transfer substrate in the

absence of detectable T pilus production. The isolation of

such mutations firmly establishes that the wild-type T pilus

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234230

is not an obligatory organelle for T4SS-mediated substrate

transfer.

The T pilus filament extending from the cell surface thus

probably functions predominantly or exclusively to bring

cells into close apposition for formation of the mating

junction. As shown in Fig. 6, this can be achieved in two

ways. Conjugative pili elaborated by the F T4SS and related

systems are thought to use an extension–retraction mecha-

nism. This mechanism is reminiscent of that described for

type IV pili produced by T2SS (as noted above, in spite of

the nomenclature, type IV pili are evolutionarily unrelated

to conjugal pili) [2,111]. The T pili assemble and establish

contact with recipient cells and then retract to bring the cells

together, most probably by an energy-dependent process.

During retraction, the pilin monomers might reinsert into the

donor cell membrane, forming a pool for use in a new round

of pilus biogenesis, or be shunted to a degradation pathway.

Once cell membranes establish direct contact, formation of

the stable mating junction triggers opening of the secretion

channel for substrate passage across the OM.

Alternatively, conjugative pili encoded by plasmids of

the IncP (e.g., RP4), IncN (pKM1010), or IncW (R388)

incompatibility groups probably do not retract, but instead

are sloughed from the cell surface (Fig. 6) [2,59]. Sloughing

can be so extensive that cell-bound pili are difficult to detect

by microscopy, as is the case for the RP4 pilus. It is not

known what signals induce pilus sloughing, but the abun-

dant accumulation of adhesive filaments in the milieu

probably induces formation of cell aggregates and, conse-

quently, mating junctions. As might be expected, cells

elaborating F-like retracting pili transfer substrates efficient-

Fig. 6. Conjugative pili likely function by a retraction mechanism, e.g., F

pilus, or a sloughing mechanism, e.g., RP4, R388, VirB/D4 pili. Transfer

systems utilizing the latter mechanism shed their pili presumably to induce

aggregation of donor and recipient cells. Both mechanisms bring donor and

recipient cell membranes into close apposition for formation of mating

junctions. Contact between donor and recipient membranes stimulates

channel opening and substrate transfer. Following transfer, the mating

junction disassembles by an unknown mechanism(s). ‘‘Uncoupling’’

mutations of the VirB/D4 T4SS permit substrate transfer on solid surfaces

in the absence of detectable pilus production [80,81].

ly in broth and on solid surfaces, whereas those dependent

on pilus sloughing systems transfer substrates efficiently

only on solid surfaces. The A. tumefaciens VirB/D4 T4SS

likely use the pilus sloughing mechanism (Figs. 5 and 6), as

suggested by extensive sequence relationships with the

IncN, IncP, and IncW conjugation systems, efficient DNA

transfer only on solid surfaces, and studies showing that T

pili accumulate at abundant levels in the extracellular milieu

[81,93].

Finally, it is interesting to consider why the VirB/D4

T4SS system—pilus and secretion channel—assembles at

the A. tumefaciens poles (Fig. 3). With respect to the

mechanics of secretion (Fig. 5) or formation of the mating

junction (Fig. 6), it is not immediately obvious why such a

polar localization would be advantageous. However, a

recent study showed that the Ti plasmid, the source of the

oncogenic T-DNA substrate, partitions to A. tumefaciens

poles during cell division [112]. This finding raises the

interesting, though completely uncharacterized, general

question of how cells coordinate two metabolic reac-

tions—plasmid partitioning and conjugation—that clearly

cannot proceed simultaneously. It is conceivable that A.

tumefaciens positioned the VirB/D4 T4SS at the poles in

order to achieve temporal and spatial control of Ti plasmid

segregation to daughter cells and T-DNA transfer to plant

cells. Intriguingly, the A. tumefaciens VirC1 protein, which

is thought to function as a Dtr protein by binding adjacent to

the T-DNA border repeats, is a ParA homolog. ParA

proteins function in segregation of chromosome and plas-

mid DNA to daughter cells during cell division [13]. Thus,

VirC1 might function as part of a global regulatory mech-

anism operating to control the timing of T-DNA transmis-

sion during the division cycle.

10. Summary and future directions

Conjugation machines are remarkably dynamic surface

organelles. Detailed structure–function studies of the A.

tumefaciens VirB/D4 T4SS and other paradigmatic conjuga-

tion systems have shown that the conjugation systems are

composed of three protein subcomplexes, Dtr, CP, and Mpf,

that act sequentially to drive three distinct reactions, sub-

strate processing, recruitment, and transfer. Two especially

noteworthy recent advances include: (i) crystallographic

structures of three T4SS subunits, homologs of the VirD4

CP, pilus-associated VirB5, and the VirB11 ATPase; and (ii)

definition of subunit–subunit contacts among machine com-

ponents. The X-ray structures and accompanying biochem-

ical findings suggest the energetic components undergo

cycles of ATP-dependent conformational changes that affect

intersubunit contacts. The TrwBR388 and HP0525Cag are

ring-shaped homohexamers structurally similar to proteins

that move along DNA and AAA ATPase unfoldase proteins,

respectively. It is tempting to suggest that ATP-binding

regulates T-strand-relaxase substrate interactions and associ-

P.J. Christie / Biochimica et Biophysica Acta 1694 (2004) 219–234 231

ated translocase or unfoldase activities. Our knowledge of

the CP and VirB11-like proteins is now sufficient to apply

structure-based mutagenesis strategies to define specific

contributions of individual residues or domains to machine

biogenesis and substrate transfer.

Correspondingly, studies defining topologies and sub-

unit–subunit contacts among the channel components are

yielding a structural view of a secretory apparatus that so far

has eluded detection by electron microscopy. The findings

have identified several interactions of interest, for example,

between energetic components, the putative IM channel

subunits VirB6, VirB8, and VirB10, and the periplasmic/

OM proteins VirB2, VirB7, and VirB9. Further studies

should address the following questions:

(1) How do the three putative ATPase subunits, VirD4,

VirB11, and VirB4, coordinate their activities to recruit

and drive substrate transfer across the IM?

(2) What is/are the IM translocase—VirD4, VirB11, or

VirB6/VirB8/VirB10—or a combination of these com-

ponents?

(3) What is the role of the VirB10 IM-OM ‘‘bridge’’ in

mediating T4SS assembly or function?

(4) What is the structure–function relationship between the

T-pilus and the secretion channel?

(5) What gating mechanisms regulate channel opening and

closure at both the IM and OM?

Over the course of the next few years, studies of these

and other questions will supply many examples—at an

increasingly atomic level of resolution—of the dynamic

nature of these fascinating translocation machines.

Acknowledgements

I thank members of my laboratory for helpful discussions

and contributions to development of the figures. I thank

Alain Bernadac for the electron micrograph showing T pili

at the A. tumefaciens cell pole. Work in my laboratory is

supported by NIH grant GM47846.

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