Download Tying rings for sex

382
33
34
35
36
37
38
39
40
Review
TRENDS in Microbiology Vol.10 No.8 August 2002
vaccine design. Clin. Diagn. Lab. Immunol.
3, 444–450
Zhu, P. et al. (2001) Fit genotypes and escape
variants of subgroup III Neisseria meningitidis
during three pandemics of epidemic meningitis.
Proc. Natl. Acad. Sci. U. S. A. 98, 5234–5239
Levin, B.R. and Bull, J.J. (1994) Short-sighted
evolution and the virulence of pathogenic
microorganisms. Trends Microbiol. 2, 76–81
Taha, M-K. et al. (2001) Circumvention of herd
immunity during an outbreak of meningococcal
disease could be correlated to escape mutation in
the porA gene of Neisseria meningitidis.
Infect. Immun. 69, 1971–1973
Derrick, J.P. et al. (1999) Structural and
evolutionary inference from molecular variation
in Neisseria porins. Infect. Immun. 67, 2406–2413
Jelfs, J. et al. (2000) Sequence variation in the
porA gene of a clone of Neisseria meningitidis
during epidemic spread. Clin. Diagn. Lab.
Immunol. 7, 390–395
Hobbs, M.M. et al. (1998) Recombinational
reassortment among opa genes from ET-37
complex Neisseria meningitidis isolates of diverse
geographical origins. Microbiology 144, 157–166
Achtman, M. (1994) Clonal spread of serogroup A
meningococci: a paradigm for the analysis of
microevolution in bacteria. Mol. Microbiol.
11, 15–22
Achtman, M. et al. (2001) Molecular epidemiology
of serogroup A meningitis in Moscow, 1969 to
1997. Emerg. Infect. Dis. 7, 420–427
41 Nicolas, P. et al. (2001) Clonal expansion of
sequence type (ST-)5 and emergence of ST-7 in
serogroup A meningococci, Africa. Emerg. Infect.
Dis. 7, 849–854
42 Swartley, J.S. et al. (1997) Capsule switching of
Neisseria meningitidis. Proc. Natl. Acad. Sci.
U. S. A. 94, 271–276
43 Kriz, P. et al. (1999) Microevolution through DNA
exchange among strains of Neisseria meningitidis
isolated during an outbreak in the Czech
Republic. Res. Microbiol. 150, 273–280
44 Kertesz, D.A. et al. (1998) Serogroup B,
electrophoretic type 15 Neisseria meningitidis in
Canada. J. Infect. Dis. 177, 1754–1757
45 Popovic, T. et al. (2000) Neisseria meningitidis
serogroup W135 isolates from U.S. travelers
returning from Hajj are associated with the ET-37
complex. Emerg. Infect. Dis. 6, 10–11
46 Taha, M-K. et al. (2000) Serogroup W135
meningococcal disease in Hajj pilgrims. Lancet
356, 2159
47 Mayer, L.W. et al. (2002) Outbreak of W135
meningococcal disease in 2000: not emergence of
a new W135 strain, but clonal expansion within
the ET-37 complex. J. Infect. Dis.
185, 1596–1605
48 Guibourdenche, M. et al. (1996) Epidemics of
serogroup A Neisseria meningitidis of subgroup
III in Africa, 1989–1994. Epidemiol. Infect.
116, 115–120
49 Kwara, A. et al. (1998) Meningitis caused by a
serogroup W135 clone of the ET-37 complex of
50
51
52
53
54
55
56
57
Neisseria meningitidis in West Africa. Trop. Med.
Int. Health 3, 742–746
Taha, M-K. et al. (2002) Neisseria meningitidis
serogroup W135 and A were equally prevalent
among meningitis cases occurring at the end of
the 2001 epidemics in Burkina Faso and Niger.
J. Clin. Microbiol. 40, 1083–1084
Abraham, S.N. et al. (1998) Fimbriae-mediated
host-pathogen cross-talk. Curr. Opin. Microbiol.
1, 75–81
Rieux, V. et al. (2001) Complex relationships
between acquisition of β-lactam resistance and
loss of virulence in Streptococcus pneumoniae.
J. Infect. Dis. 184, 66–72
Bayliss, C.D. et al. (2001) The simple sequence
contingency loci of Haemophilus influenzae and
Neisseria meningitidis. J. Clin. Invest.
107, 657–662
Müller-Graf, C.D.M. et al. (1999) Population
biology of Streptococcus pneumoniae isolated from
oropharyngeal carriage and invasive disease.
Microbiology 145, 3283–3293
Côté, S. et al. (1994) Molecular typing of
Haemophilus influenzae using a DNA probe and
multiplex PCR. Mol. Cell. Probes 8, 23–37
Hausdorff, W.P. et al. (2000) Which pneumococcal
serogroups cause the most invasive disease:
implications for conjugate vaccine formation and
use, part I. Clin. Infect. Dis. 30, 100–121
Zhang, Y.X. et al. (2002) Genome shuffling leads to
rapid phenotypic improvement in bacteria.
Nature 415, 644–646
Tying rings for sex
Markus Kalkum, Ralf Eisenbrandt, Rudi Lurz and Erich Lanka
The primary component of the sex pilus encoded by IncP (RP4) and Ti plasmids
has been identified as a circular pilin protein with a peptide bond between
the amino and carboxyl terminus. Here, we review the key experiments that
led to this discovery, and the present mechanistic model for pilin-precursor
processing and the cyclization reaction. In addition, we discuss the
implications for horizontal gene transfer in bacterial conjugation.
Published online: 10 July 2002
Bacterial conjugation efficiently mediates horizontal
gene transfer in a highly promiscuous manner.
Conjugative processes enable bacteria to transfer
conjugative plasmid DNA not only between members
of their own kingdom, but also to fungi, plants and
even mammalian cells, as recent laboratory
experiments have indicated [1]. The secretion of
macromolecules via bacterial conjugation is
categorized as a type IV secretion process
(recently reviewed by Christie and Vogel [2]).
The initial step in bacterial conjugation requires
physical contact between the donor ‘male’ and recipient
‘female’ cells. The conjugative plasmid DNA in the
donor cell is relaxed at the origin of transfer (oriT) by
http://tim.trends.com
proteins belonging to the DNA transfer and relaxation
(DTR) system, then channelled into the periplasm
through the lumen of a hexameric protein. This
structure is formed by the TraG protein in RP4mediated conjugation and by its homologue TrwB in
R388 (IncW)-mediated conjugation [3]. Mating pair
formation (Mpf) proteins, which span the cell envelope,
are required to transfer the plasmid DNA into the
recipient cell [4] (Fig. 1). Electrophysiological studies
have shown that the presence of Mpf proteins enhances
the permeability of the host cell envelope [5]. The
mechanistic details of the transfer of DNA and other
macromolecules and the presumed accessory functions
of Mpf proteins are currently being investigated.
The 3-D structure of the Helicobacter pylori Cagα
protein, a member of the VirB11 protein family of
‘traffic’ nucleoside triphosphatases, was solved
recently [6]. Three proteins belonging to this group,
TrbB (encoded by the RP4 plasmid), Cagα (H. pylori),
and TrwD [encoded by the R388 (IncW) plasmids],
have been shown to form homohexameric rings with a
central hole [7]. TrbB, Cagα and TrwD are cytoplasmic
proteins and are associated with the inner membrane.
0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(02)02399-5
Review
TRENDS in Microbiology Vol.10 No.8 August 2002
Phages
Recipient cell
Pf3
PRR1
PRD1
Entrance
of phage
DNA/RNA
?
Interaction
with envelope
Pilin
(cyclic TrbC)
?
Detached pilus
detected by EM
Mpf
proteins
A
RP4
plasmid
TraG
ss D N
DTR
proteins
TrbC processing
TraF
Pilin precursors
(linear TrbC)
Donor cell
TRENDS in Microbiology
Fig. 1. The RP4 pilus in bacterial conjugation. It is proposed that the pilus is assembled from circular
TrbC subunits by proteins of the envelope-spanning Mpf system. A rudimentary pilus ‘stump’ might
exist near Mpf proteins on the cell surface. It can be recognized by specific phages that take over
functionalities of the host’s conjugative apparatus for the purpose of their own propagation. The
host-cell’s RP4 plasmid is relaxed, nicked and transferred into the periplasm by the DNA transfer and
relaxation (DTR) apparatus. Relative dimensions within the drawing are based on actual sizes.
Rudi Lurz
Erich Lanka*
Max-Planck-Institut für
Molekulare Genetik,
D-14195 Berlin, Germany.
*e-mail:
[email protected]
Markus Kalkum
Dept of Mass
Spectrometry and
Gaseous Ion Chemistry,
The Rockefeller
University,
New York, NY 10021-6399,
USA.
Ralf Eisenbrandt
Biochemie GmbH,
Biochemiestrasse 10,
A-6250 Kundl, Austria.
The delivery of effector molecules through the
barrier formed by four bacterial membranes occurs
following the formation of thin, tube-like extracellular
filaments, the conjugative pili. It remains an open
question whether DNA or other macromolecules per se
are transported through the pili. For IncP pili, their
brittle structure and loose attachment to the bacterial
surface does not suggest such a role (discussed later).
Although suggested by its analogy to the F pilus,
evidence that the RP4 pilus is erect on the host cell
and spans the gap to the recipient cell remains
elusive. In fact, electron micrographs of
RP4-containing host cells show the pili to be detached
from the cell surface. So far, the best hint for the
existence of a residual pilus ‘stump’ on the cell surface
comes from studies of bacteriophages that bind
specifically either to the pilus or to other Mpf proteins
(Fig. 1). The basic structure of the pilus associated
with IncF, IncP and Ti plasmids consists of a single
type of pilin subunit. The IncP and Ti pilins are cyclic
proteins whose amino and carboxyl termini are joined
by a peptide bond. A series of proteolytic truncations
of the initially linear precursors lead to the formation
of this covalent head-to-tail linkage. This review will
focus on the maturation of pilin precursors in
conjugation and type IV secretion systems.
Pilus structure and function
The model conjugative pilus is the F pilus, a thin
filament attached to the surface of conjugative
F+ bacteria [8]. The F pilus is a tubular structure of
8.5–9 nm in diameter with an inner axial hole that is
2–2.5 nm in diameter. The filament is flexible, varies in
http://tim.trends.com
383
length between 1 and 2 µm and is composed of multiple
copies of a single subunit. This subunit, known as pilin,
is an N-acetylated, 71-residue polypeptide encoded
by the traA gene [9]. The proposed role of the pilus in
conjugation is to establish physical contact between
the donor and recipient cells; this contact is initiated
when the donor cell attaches the tip of the pilus to the
recipient cell. A depolymerization step is thought to
pull donor and recipient together, thus allowing the cell
envelopes to engage in intimate contact [8]. These
mating aggregates are thought to facilitate the
transmission of single-stranded (ss) DNA through the
barrier formed by the two cell envelopes of mating
Gram-negative cells, which comprises four membranes
and two dense peptidoglycan networks, and has a total
thickness of ~500 Å.
Other conjugation systems also encode pili.
Perhaps the most comprehensive survey on
conjugative pili, in which the pili were characterized
mainly by morphological and immunological aspects,
was that of Bradley [10]. The F-transfer system shows
high mating efficiency in liquid with considerably
lower efficiency on semi-solid media, whereas IncP
plasmids are known for high mating efficiencies on
semi-solid media and low efficiencies in liquid. The
number of pili on F+ cells is approximately two to three
per cell. By contrast, only one out of 50 E. coli cells
containing the IncP plasmid RP4 is visibly piliated.
The existence of the rigid IncP pili was visualized more
convincingly in cell preparations that overproduced
the mating pair apparatus. On the RP4 plasmid, the
20 essential transfer genes [11] are located in two
isolated regions, Tra1 and Tra2. The DNA transfer
and replication functions are encoded by Tra1 whereas
IncP pilus biogenesis requires one transfer gene from
the Tra1 cluster and ten Tra2 genes. These latter
11 genes are responsible for the assembly of the
proposed supramolecular Mpf complex [4] (Fig. 1). As
each type IV secretion [2] gene cluster contains a
potential pilin precursor (prepilin) gene, the recently
discovered phylogenetic and functional relationship
between the IncP mating pair apparatus and type IV
secretion systems of mammalian and plant pathogens
might help to dissect the pilus assembly process.
Investigation of the pilus assembly pathway began
with the identification of the RP4 (IncPα) prepilin
gene and determination of the chemical nature of the
pilus subunit.
A characteristic of IncP pili is their tendency to
aggregate in bundles (Fig. 2); the hydrophobic surfaces
of the pili are probably responsible for interactions
between the filaments. On some filaments a fine
longitudinal middle line suggests a tubular structure
for the pilus. Occasionally, filaments with a diameter
of ~10 nm display a ‘knob’ at one end, like a small
sacculus [12,13], which might indicate a former
anchor in the outer membrane of the cell envelope.
Surprisingly, inspection of cells producing pili in high
quantities shows that the majority were detached from
the cell surface and arranged in bundles resembling
Review
384
TRENDS in Microbiology Vol.10 No.8 August 2002
Fig. 2. Electron
micrograph of the RP4
pilus. Scale bar = 200 nm.
the behaviour of purified preparations. Thus,
hydrophobic cell-to-cell interactions could be facilitated
by the pili supporting adherence and the formation
of mating aggregates. To date, there is still no
experimental evidence to support the idea that genetic
material is transported through the pilus itself.
The RP4 model system
Broad host range plasmids belonging to the IncP
family specify resistance to multiple antibiotics
including kanamycin, gentamicin, tetracycline,
penicillin, streptomycin, sulfonamide,
chloramphenicol and trimethoprim, and resistance to
mercury ions [14]. IncP plasmids are found in hosts
(Gram-positive and -negative) that are spread
ubiquitously, but particularly in surroundings with a
high selective pressure, including hospitals, animal
husbandry premises and fish farms. As bacterial
conjugation is the major pathway for the spread of
resistance genes [15], IncP plasmids such as RP4 have
served as a model system to study DNA transmission
in the environment and at the molecular level [16,17].
Signal
peptide
PreProTrbC
Host
peptidase
36 aa
TMH
TMH
27 aa
LepB
ProTrbC
TraF
4 aa
TrbC*
TraF
Pilin
78 aa
TRENDS in Microbiology
Fig. 3. Maturation cascade of the RP4 pilin. TrbC, 145 residues in length, is represented by a bar. Defined
sections of TrbC are marked: signal peptide (red), core region (light green), trans-membrane helices
(TMH, dark green), carboxy-terminal end (light blue) and tetrapeptide (dark blue).
http://tim.trends.com
One recent achievement was the unambiguous
immunological identification of the trbC gene product
as the pilus precursor [12]. The existence of different
forms of TrbC indicated a complex process of
modification of the 145-residue TrbC polypeptide
[18,19] (Fig. 3). The precursor maturation cascade
begins with removal of a 27-residue polypeptide from
the carboxyl terminus by an as-yet-unidentified
host-encoded protease. As a second distinct step, the
36-residue signal peptide is cleaved off by the hostencoded signal peptidase LepB of E. coli. The third
and final modification step was revealed by mass
spectrometry (see below). This step yields the mature
cyclic pilin that makes up the pilus: truncation of the
carboxyl terminus by four residues and the formation
of a new peptide bond connecting the amino and
carboxyl terminus of TrbC in one concerted reaction.
The cyclization is catalyzed by the essential plasmidborne transfer protein, TraF, which has sequence
similarity to signal peptidases [18]. TraF homologues
have been shown to belong to a special class of serine
proteases [20]: their catalytic activity results from
serine–lysine dyad formation. Mutation of Ser37 and
Lys89 of TraF reduced the activity of the protein, which
did not support the synthesis of conjugative pili [18]. We
propose a mechanism based on the action of serine
proteases (Fig. 4). The carboxyl group of Gly78 of the
membrane-anchored and doubly processed TrbC is
proposed to react and form an acyl-intermediate with
Ser37 of TraF. Nucleophilic attack of the Ser37 hydroxyl
group requires preemptive activation by a dyad-like
deprotonation via Lys89 or through other possible
proton acceptors. In fact, Asp155, which is essential
for the enzymatic function of TraF [19], might even be
involved in the activation step. The carboxy-terminal
tetrapeptide (AEIA) is cleaved off while the TrbC acylenzyme complex undergoes aminolysis by reacting with
the amino terminus of TrbC. The existence of two
transmembrane hydrophobic helices (TMH, see
Fig. 3) in TrbC supports our mechanistic model. They
ensure that both the amino and carboxyl terminus of
TrbC are localized at the same side of the membrane,
presumably in the periplasm. The formation of a
new peptide bond between the carboxyl and amino
terminus seems to use conserved energy from the
removal of the tetrapeptide. A conceivable alternative
reaction – the hydrolysis of the TraF-acyl-TrbC
intermediate and the resulting truncated,
non-circular form of TrbC – was not observed. None
of the mutant proteins used in mechanistic studies
displayed loss of the tetrapeptide without concerted
cyclization of the residual TrbC [19], indicating that
the removal of the tetrapeptide cannot be uncoupled
from the cyclization reaction.
A thorough analysis of the chemical nature of
the RP4 pilus and pilin was performed using
matrix-assisted laser desorption/ionization–time of
flight-mass spectrometry (MALDI–TOF-MS) [12].
Preparations of purified pili were subjected to different
matrices to find the optimal conditions for sensitive
Review
TRENDS in Microbiology Vol.10 No.8 August 2002
Fig. 4. Proposed
mechanism of the pilin
cyclization catalyzed by
TraF on the periplasmic
side of the cytoplasmic
membrane.
To proton
acceptor
385
Leaving tetrapeptide
H
+
O
NH2
O=
O
NH2
C
TrbC
No hydrolysis
detected
O=C
TraF
Aminolysis
Pilus
assembly
H
HO
+
HN
O
C
O-
TRENDS in Microbiology
detection of pilin ions. Of a variety of matrices tested,
trans-3-indole acrylic acid (IAA) yielded the most
intense signals for the pilin or its precursors (Fig. 5).
IAA permitted us to detect intense pilin signals even
from matrix preparations with whole E. coli or
Agrobacterium tumefaciens cells. The choice of IAA as
the MALDI-MS matrix proved to be key to the
analysis of a variety of TrbC and TraF mutants.
The circular nature of the mature pilin was
deduced from mapping tryptic and chymotryptic
proteolysis products in MALDI-MS experiments.
Although pili resist protease treatment to a great
extent, enough material was digested when pilus
suspensions were exposed to high concentrations
of the enzymes [12]. A time course of the tryptic
digestion of pilin monitored by mass spectrometry
revealed another feature unique to circular proteins:
the molecular mass increases by 18 daltons when the
first peptide bond is hydrolyzed – consistent with the
formal addition of one water molecule.
Sensitive mass measurements of pilins from entire
cells in the IAA matrix allowed us to investigate a
variety of IncP pilin mutations reliably without the
need for elaborate pilus purification. Future studies
on other pilins should take into account that it might
not always be possible to find a suitable matrix that
http://tim.trends.com
discriminates against background proteins as IAA
does for the pilins of the IncP and IncRH1 plasmids.
Different physico-chemical properties and expression
levels must also be considered.
Analogies to related systems
Biogenesis of the T pilus occurs upon expression of the
virB operon of the Ti plasmid with a processed form of
VirB2 being assembled into the T pilus. Our studies on
pilin maturation in the Vir system revealed that an
enzyme encoded by the chromosome is required for the
cyclization reaction of VirB2 [21]. It was found that
VirB2 becomes cyclized in A. tumefaciens strains
but not in E. coli [21] and that VirB2 propilin was
processed and cyclized in the absence of any other
Ti plasmid gene [12,21]. Except for a weak sequence
similarity between TraF and VirF/Orf2 of the
Ti plasmid, which has been shown to be dispensable
for conjugative transfer [22], the Ti plasmid lacks a
functional TraF homologue. Thus, the enzyme for
Ti propilin cyclization appears to be of chromosomal
origin. In addition to VirB2, the VirB5 protein is a
putative component of the T pilus and co-fractionates
with T pilus preparations. Although VirB2 is the major
pilus subunit, VirB5 might be directly involved in
T pilus assembly, possibly as a minor component [13]
Review
Fig. 5. The effect of
matrices on the signal
intensities of pilin ions in
preparations with entire
bacteria. The amount of
bacteria is the same in all
three matrix-assisted laser
desorption/ionizationmass spectrometry
(MALDI-MS) spectra.
Trans-3-indole acrylic acid
(IAA) discriminates
strongly against other
Escherichia coli proteins.
2-(4-hydroxyphenyl azo)benzoic acid (HABA)
yields little discrimination
and sinapic acid
(3-(4-hydroxy-3,5dimethoxy phenyl) acrylic
acid) (SA) shows no
usable gain in the
intensity of the pilin signal
at 8119.6 m/z. Other
commonly used matrices
like 4-hydroxy-α-cyano
cinnamic acid and
2,4-dihydroxy benzoic
acid gave broader and
less intense signals
(not shown).
TRENDS in Microbiology Vol.10 No.8 August 2002
8119,6
Matrix
O
OH
Relative intensity
386
IAA
NH
O
OH
HABA
N N
OH
O
O
OH
HO
SA
O
7000
9000
m/z
TRENDS in Microbiology
and perhaps as a chaperone [23]. A second transfer
system of the Ti plasmid, responsible for conjugation
between A. tumefaciens cells, consists of three
Tra regions [24]. The Mpf system encoded by the
Ti plasmid contains highly conserved TraF and TrbC
homologues. The RP4 TrbC cyclization motif (G/AIEA)
(Fig. 6) was defined by mutagenesis. A similar motif
(G/AISG) is present in TrbC of the Ti plasmid and we
therefore propose that, analogous to the IncP system,
the conjugative Ti pilin is a cyclic polypeptide [12].
VirB2
PtIA
TrbC Ti
TrbC
28 AIEPNLAHANGG
: : || | :|
25 ATLPDLAQAGGG
: :::|| | |
20 IGLADPAFASSG
: : | | |
28 ALSAHPAMASEG
39 ...
77 GAAAEIASYLL 87
:: |:|: :::
36 ... 74 GASAEIARYLL 84
: |:|:
31 ... 103 ATGASIGEMEA 113
: |:|:
39 ... 96 GRGAEIAALGN 106
TRENDS in Microbiology
Fig. 6. Putative cyclization motifs. Partial sequence comparison of RP4 TrbC (M93696), Ti TrbC
(P54908), Bordetella pertussis PtlA (L10720), Brucella suis VirB2 (AF141604) and Brucella abortus
VirB2 (AF226278). The two VirB2 sequences are identical and are shown once only. GenBank
accession numbers are given in parenthesis. The first and last amino acid position in each line is
numbered according to the full-length proteins. Lines correspond to conserved residues in all, colons
to residues conserved in at least two sequences. Residues of a proposed common maturation process
are highlighted in green. Arrows mark the sites of RP4 TrbC processing.
http://tim.trends.com
In some macromolecular secretion systems of
mammalian pathogens, cyclic peptides apparently
play a crucial role. The pertussis toxin operon of
Bordetella pertussis [25] encodes the pilin-like protein
PtlA and two related virulence operons of Brucella
suis [26] and Brucella abortus [27] are proposed to
contain virB2 genes that encode pilus subunits [19]
(Fig. 6). Each of the three sequences displays high
sequence similarity to the potential processing motifs
of TrbC [19]. Predicted pilin precursors from plasmids
R6K (PilX2), R388 (TrwL), pKM101 (TraM) and
HP0546 of H. pylori (Werner Pansegrau, pers.
commun.) are homologous to TrbC and VirB2. The
precursors for the putative pilins are hydrophobic
polypeptides, each <150 residues in size. They contain
amino-terminal signal peptides of considerable length
(25–50 residues) and two predicted transmembrane
helices in the core region. A common feature shared
by these proteins is their indifferent behavior to
most protein dyes (Coomassie, Sypro Orange® and
Amido Black). These dyes do not stain the proteins
in polyacrylamide SDS gels and so far, only silver
staining has been successfully applied to detect
separated bands of these proteins [28]. Furthermore,
it was found that strains overproducing the respective
pilus subunit terminate cell growth and division
immediately after expression is induced (C. Rabel and
E. Lanka, unpublished). Hence, nutrient uptake by
these cells might be hampered by the membranetargeting pilin, suggesting possible interference or
inhibition of essential transport mechanisms through
the inner membrane.
Cyclic proteins
The formation of cyclic pilus subunits during sex
pilus biogenesis is a recently discovered process. The
implications for the function and biological relevance
of other cyclic proteins remain largely unknown.
Several biologically active cyclic peptides exist and
can be assigned to either one of two groups: first,
small cyclic peptides that originate from a complex
enzymatic pathway similar to fatty acid metabolism
[29,30], and that usually display ion-chelating and
antibiotic activity. Examples include Gramicidin-S,
actinomycin, polymyxin, tyrocidin and etamycin.
Second, a small group of ribosomally synthesized
proteins, among them the cyclic antibiotic AS-48 from
Enterococcus faecalis [31], which is inserted into the
membrane of the target cell and causes proton loss
owing to permeabilization of the membrane. The
precise mechanism involved and the possible catalytic
function responsible for the cyclization of AS-48
remains to be discovered. Other peptides that
undergo cyclization via disulfide bonds rather than
peptide bonds (such as somatostatin) will not be
addressed here. Engineered cyclic proteins with
peptide bonds in head-to-tail linkages have been
reported [32,33]; backbone cyclization was achieved
using a modified version of protein splicing that
involves intein technology.
Review
TRENDS in Microbiology Vol.10 No.8 August 2002
Uninvited guests
Acknowledgements
We thank Hans Lehrach
for generous support.
Work in E.L.’s laboratory
was supported by
the Deutsche
Forschungsgemeinschaft.
The EU-BIOTECH
concerted action BIO4-CT0099, Mobile genetic
Elements’ Contribution to
Bacterial Adaptability and
Diversity (MECBAD)
provided a platform for
fruitful discussions.
The acquisition of additional genetic information
is not always an advantage. Although bacterial
resistance to antibiotics makes the presence of
conjugative plasmids and the formation of conjugal
pili advantageous features in certain environments,
some ‘uninvited guests’ use these extracellular
appendices as a receptor to attach, enter and destroy
their hosts. So-called donor, or male-specific, bacterial
viruses attach to pili. Viruses PRR1 (RNA),
Pf3 (ssDNA), and PRD1 (double-stranded DNA)
use RP4-containing cells as preferred targets, using
one of two strategies. Whereas Pf3, a filamentous
phage, and PRR1, an icosahedral phage, attach along
the pilus, PRD1 adsorption is only detected on the
surface of RP4-containing cells [19]. All three types
of phages need the 11 Mpf core functions that are
required for conjugation, including a functional cyclic
pilin, for their own propagation [34,35]. The Mpf
components are membrane proteins likely to form the
pilus assembly apparatus by bridging the inner and
outer membrane [4]. Thus, the cyclic protein TrbC
plays a crucial role not only during conjugation
References
1 Waters, V.L. (2001) Conjugation between bacterial
and mammalian cells. Nat. Genet. 29, 375–376
2 Christie, P.J. and Vogel, J.P. (2000) Bacterial
type IV secretion: conjugation systems adapted to
deliver effector molecules to the host cells.
Trends Microbiol. 8, 354–360
3 Gomis-Ruth, F.X. et al. (2002) Conjugative
plasmid protein TrwB, an integral membrane
type IV secretion system coupling protein.
Detailed structural features and mapping of the
active site cleft. J. Biol. Chem. 277, 7556–7566
4 Grahn, A.M. et al. (2000) Components of the RP4
conjugative transfer apparatus form an envelope
structure bridging inner and outer membranes of
donor cells: implications for related
macromolecule transport systems. J. Bacteriol.
182, 1564–1574
5 Daugelavi?ius, R. et al. (1997) The IncP plasmidencoded cell envelope-associated DNA transfer
complex increases cell permeability. J. Bacteriol.
179, 5195–5202
6 Yeo, H-J. et al. (2000) Crystal structure of the
hexameric traffic ATPase of the Helicobacter pylori
type IV secretion system. Mol. Cell 6, 1461–1472
7 Krause, S. et al. (2000) Sequence related protein
export NTPases encoded by the conjugative
transfer region of RP4 and by the cag
pathogenicity island of Helicobacter pylori share
similar hexameric ring structures. Proc. Natl.
Acad. Sci. U. S. A. 97, 3067–3072
8 Achtman, M. et al. (1978) Cell–cell interactions in
conjugating Escherichia coli: role of F-pili and fate
of mating aggregates. J. Bacteriol. 135, 1053–1061
9 Frost, L.S. et al. (1986) Two monoclonal antibodies
specific for different epitopes within the aminoterminal region of F pilin. J. Med. Microbiol.
168, 192–198
10 Bradley, D.E. (1980) Morphological and serological
relationship of conjugative pili. Plasmid 4, 155–169
11 Lessl, M. et al. (1992) Dissection of IncP
conjugative plasmid transfer: definition of the
transfer region Tra2 by mobilization of the Tra1
region in trans. J. Bacteriol. 174, 2493–2500
http://tim.trends.com
387
(DNA export from the cell) but also in phage DNA
transfer (DNA transfer into the cell). It is already
possible to find mutations in trbC that can dissect
these two processes [19] but further studies will be
needed to propose a detailed mechanism.
The future
The architecture of the pilus assembly machinery,
the functions and interactions of the components in
the complex and the crystal structure of the pilus
are challenging projects to be addressed. How is
contact established between the pilus and the
recipient cell surface? Are there specific pilus
receptors or do cyclic TrbC molecules enhance the
permeability of the recipient cell wall? In silico data
suggest several functional parallels between
conjugative transfer systems and type IV secretion
pathways. One of the intriguing questions is whether
type IV systems produce pili or whether the proposed
pilin precursors, such as PtlA (B. pertussis) and
0546 (H. pylori), have other roles in the secretion
processes of bacterial pathogens. Continued research
is therefore required.
12 Eisenbrandt, R. et al. (1999) Conjugative pili of IncP
plasmids, and the Ti plasmid T pilus are composed
of cyclic subunits. J. Biol. Chem. 274, 22548–22555
13 Schmidt-Eisenlohr, H. et al. (1999) Vir proteins
stabilize VirB5 and mediate its association with
the T pilus of Agrobacterium tumefaciens.
J. Bacteriol. 181, 7485–7492
14 Smith, D.I. et al. (1975) Third type of gentamicin
resistance in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother. 8, 227–230
15 Waters, V.L. (1999) Conjugative transfer in the
dissemination of beta-lactam and aminoglycoside
resistance. Front. Biosci. 4, D433–D456
16 Dröge, M. et al. (2000) Phenotypic and molecular
characterization of conjugative antibiotic resistance
plasmids isolated from bacterial communities of
activated sludge. Mol. Gen. Genet. 263, 471–482
17 Guiney, D.G. and Lanka, E. (1989) Conjugative
transfer of IncP plasmids. In Promiscuous
Plasmids of Gram-negative Bacteria
(Thomas, C.M., ed), pp. 27–56, Academic Press
18 Haase, J. and Lanka, E. (1997) A specific protease
encoded by the conjugative DNA transfer system
of IncP and Ti plasmids is essential for pilus
synthesis. J. Bacteriol. 179, 5728–5735
19 Eisenbrandt, R. et al. (2000) Maturation of
IncP-pilin precursors resembles the catalytic
dyad-like mechanism of leader peptidases.
J. Bacteriol. 182, 6751–6761
20 Paetzel, M. and Dalbey, R.E. (1997) Catalytic
hydroxyl/amine dyads within serine proteases.
Trends Biochem. Sci. 22, 28–31
21 Lai, E.M. et al. (2002) Biogenesis of T pili in
Agrobacterium tumefaciens requires precise
VirB2 propilin cleavage and cyclization.
J. Bacteriol. 184, 327–330
22 Melchers, L.S. et al. (1990) Octopine and nopaline
strains of Agrobacterium tumefaciens differ in
virulence; molecular characterization of the virF
locus. Plant Mol. Biol. 14, 249–259
23 Lai, E.M. and Kado, C.I. (2000) The T-pilus of
Agrobacterium tumefaciens. Trends Microbiol.
8, 361–369
24 Li, P.L. et al. (1998) Genetic and sequence analysis
25
26
27
28
29
30
31
32
33
34
35
of the pTiC58 trb locus, encoding a mating pair
formation system related to members of the type
IV secretion family. J. Bacteriol. 180, 6164–6172
Weiss, A.A. et al. (1993) Molecular characterization
of an operon required for pertussis toxin secretion.
Proc. Natl. Acad. Sci. U. S. A. 90, 2970–2974
O’Callaghan, D. et al. (1999) A homologue of the
Agrobacterium tumefaciens VirB and Bordetella
pertussis Ptl type IV secretion systems is essential
for intracellular survival of Brucella suis.
Mol. Microbiol. 33, 1210–1220
Sieira, R. et al. (2000) A homologue of an operon
required for DNA transfer in Agrobacterium is
required in Brucella abortus for virulence and
intracellular multiplication. J. Bacteriol.
182, 4849–4855
Eisenbrandt, R. (1999) Macromolecular Export
Systems, Identification of Conjugative Pilins and
Their Modification, Logos Verlag, Berlin.
Saito, Y. and Otani, S. (1970) Biosynthesis of
gramicidin S. Adv. Enzymol. Relat. Areas Mol. Biol.
33, 337–380
Kleinkauf, H. and von Dohren, H. (1990)
Nonribosomal biosynthesis of peptide antibiotics.
Eur. J. Biochem. 192, 1–15
Samyn, B. et al. (1994) The cyclic structure of the
enterococcal peptide antibiotic AS-48. FEBS Lett.
352, 87–90
Iwai, H. et al. (2001) Cyclic green fluorescent
protein produced in vivo using an artificially split
PI-PfuI intein from Pyrococcus furiosus.
J. Biol. Chem. 276, 16548–16554
Iwai, H. and Plückthun, A. (1999) Circularlactamase: stability enhancement by cyclizing the
backbone. FEBS Lett. 459, 166–172
Haase, J. et al. (1995) Bacterial conjugation mediated
by plasmid RP4: RSF1010 mobilization, donorspecific phage propagation, and pilus production
require the same Tra2 core components of a proposed
DNA transport complex. J. Bacteriol. 177, 4779–4791
Grahn, A.M. et al. (1997) Assembly of a functional
phage PRD1 receptor depends on 11 genes of the
IncP plasmid mating pair formation complex.
J. Bacteriol. 179, 4733–4740